What would happen to your memories and personality if, after decades of adult life, some portion of your brain was replaced with the progeny of fresh stem cells (as might happen in a treatment for degenerative brain disease)? Given the fascinating but poorly-understood examples of memory in aneural systems such as plants, ciliates, etc. (Eisenstein, 1975; Brugger et al., 2002; Volkov et al., 2008), is it possible that memories can be stored in tissues other than the brain? For that matter, how can arbitrary mental content (specific memories) be encoded and decoded within a physical medium such as a brain? What would be the dynamics of memories during brain regeneration?

It has been shown that memories survive the drastic reorganization of the nervous system during metamorphosis from larva to adult in insects (Sheiman and Tiras, 1996; Blackiston et al., 2008). However, there is only one model system in which true memory and complete brain regeneration can be studied in the same animal: planaria. Planaria are free-living flatworms with bilateral symmetry, a true centralized brain, and a very rich behavioral repertoire. They are thus a popular system for the study of addiction and withdrawal. They also have remarkable powers of regeneration: a resident population of adult stem cells (neoblasts) enable these worms to regenerate any bodypart after surgical removal (Gentile et al., 2011; Lobo et al., 2012). In the 1960’s, a visionary named James V. McConnell experimentally posed a bizarre question (McConnell, 1965): if planaria were trained to form a specific memory and then regenerated their entire brain after decapitation, would the memories still be accessible?

McConnell’s group performed a number of experiments that suggested that the answer was Yes (McConnell et al., 1959; McConnell, 1966) – animals regenerating their whole head seemed to show recall of memories formed via several different training paradigms and behavioral assays. Unfortunately, at the time, training and testing experiments had to be done by hand. Manual experiments with worms are very tedious – not only time-consuming (resulting in low N’s and weak memories in worms who can only be trained for a short time per day) but also open to subjectivity during scoring of behaviors and very hard to control (behavioral experiments are notoriously sensitive to the skill of the operator). These difficulties led to the results being confirmed in some labs but not reproduced in some others (Corning, 1967; Corning and Riccio, 1970; Travis, 1981), and the whole area was largely forgotten (Rilling, 1996; Smalheiser et al., 2001) as people moved on to genetically-tractable model systems and easier questions of neuroscience.

In our lab, we are interested in information processing in cells and tissues. We study how cells orchestrate their activity towards maintaining and repairing complex anatomies, and how behavioral programs interact with radically-altered bodyplans (Levin, 2011). Indeed, our work has shown that all cells, not just excitable nerve, can communicate via changes of resting potential during patterning in embryogenesis, regeneration, and cancer suppression (Levin, 2012). This led to the hypothesis that perhaps networks of non-neural cells might also support the memory and information-processing abilities of neural nets; perhaps the ability of organisms to detect deviations from their target morphology and effect repairs is a reflection of true memory of specific shapes implemented by non-neural bioelectrical networks (Tseng and Levin, 2013)? Our work blurs the line between cognitive science and developmental biology, and we wondered if there may be mechanistic similarities between memory of spatial pattern (morphogenesis) in somatic structures, and memory of temporal patterns (learning and inference) in the brain. Aside from understanding development and developing interventions to increase regenerative ability, we are also interested in the synthetic bioengineering of hybrid tissues with new information-processing capabilities (developing new distributed computational platforms).

In order to establish a new model for studying encoding of information in living tissues and probe the mechanisms that interface the mind to the body, we turned to the planarian. Our first step was to build a next-generation testing and training device for flatworms (it also works for Xenopus and zebrafish (Blackiston and Levin, 2012; Blackiston and Levin, 2013)). The goal was to overcome prior roadblocks for this work: our system not only tracks the movement of planaria, but uniquely provides individual feedback (rewards and punishments) to 12 worms at a time, consistently (24*7 training), thus enabling not only behavior analysis but automated training in learning paradigms (Hicks et al., 2006; Blackiston et al., 2010). The idea was to do away with variability in manual training procedures, overcome operator tedium, and produce quantitative, objective, computer-scored analysis of worm performance after training. Five years and many engineering problems later, we had a device and a successful training protocol for planaria.

Amazingly, the data showed that if worms were trained to remember a novel kind of chamber environment with a specifically rough (laser-etched) surface, they would recognize it again (as measured by their willingness to eat a piece of liver rather than spend time exploring a new environment as controls do) after complete head removal and regeneration (Shomrat and Levin, 2013). While there is still plenty of room for improving the training protocol to induce an even more robust memory, and the worms need a brief refresher after head regeneration in order to show good recall of the original training, the results clearly showed that a complex brain-derived behavior was driven by a memory that survived complete head regeneration.
Our data suggest that not only can memories be stored somewhere outside of the brain, but that they can be imprinted on a naïve regenerated brain. Our future efforts will be focused on molecular cell biology and biophysics approaches to understand 1) which tissues contain the memory (e.g., are neoblasts required? Is it everywhere, or in certain regions?), 2) what molecular pathways underlie the imprinting of this information onto the brain, and 3) how specific memories are encoded into, and decoded from, living tissues. These data establish the automated analysis of memory in regenerated planaria as a new, highly tractable, model system in which to probe new aspects of cognitive science and the intersection of neurobiology and regeneration.

At the moment, we do not know where and how the memory is encoded in the body, or how prevalent such pathways are throughout the tree of life. However, based on exciting recent work on bioelectricity in somatic cells (McCaig et al., 2005; Allen et al., 2011; Bissiere et al., 2011; Wu et al., 2011; Tseng and Levin, 2013), we suggest the fascinating possibility that memory could be globally distributed throughout the body: non-neural cells communicating bioelectrically through gap junctions (electrical synapses) form a neural-like network that could be a very rich medium for encoding information and directing cell activity during regeneration (Chakravarthy and Ghosh, 1997; Bose and Karmakar, 2003; Inoue, 2008). Future work in this system will shed light not only on fundamental issues of memory but also on the biomedically-relevant questions of how regenerative therapies interact with cognitive content in patients. There will also likely be interesting applications of hybrid or biologically-inspired technologies for computational tissues and new computer architectures (Costello et al., 2011; Holley et al., 2011; Adamatzky, 2012; Adamatzky et al., 2012) that we can yet only imagine in their vaguest form.

References Cited

    Adamatzky, A. (2012) ‘Slime mold solves maze in one pass, assisted by gradient of chemo-attractants’, IEEE transactions on nanobioscience 11(2): 131-4.
 
   Adamatzky, A., Holley, J., Dittrich, P., Gorecki, J., De Lacy Costello, B., Zauner, K. P. and Bull, L. (2012) ‘On architectures of circuits implemented in simulated Belousov-Zhabotinsky droplets’, Biosystems 109(1): 72-7.
 
   Allen, K., Fuchs, E. C., Jaschonek, H., Bannerman, D. M. and Monyer, H. (2011) ‘Gap Junctions between Interneurons Are Required for Normal Spatial Coding in the Hippocampus and Short-Term Spatial Memory’, J Neurosci 31(17): 6542-52.
 
   Bissiere, S., Zelikowsky, M., Ponnusamy, R., Jacobs, N. S., Blair, H. T. and Fanselow, M. S. (2011) ‘Electrical synapses control hippocampal contributions to fear learning and memory’, Science 331(6013): 87-91.
 
   Blackiston, D., Shomrat, T., Nicolas, C. L., Granata, C. and Levin, M. (2010) ‘A second-generation device for automated training and quantitative behavior analyses of molecularly-tractable model organisms’, PLoS ONE 5(12): e14370.
 
   Blackiston, D. J. and Levin, M. (2012) ‘Aversive training methods in Xenopus laevis: general principles’, Cold Spring Harbor Protocols 2012(5).
 
   Blackiston, D. J. and Levin, M. (2013) ‘Ectopic eyes outside the head in Xenopus tadpoles provide sensory data for light-mediated learning’, The Journal of experimental biology 216(Pt 6): 1031-40.
 
   Blackiston, D. J., Silva Casey, E. and Weiss, M. R. (2008) ‘Retention of memory through metamorphosis: can a moth remember what it learned as a caterpillar?’, PLoS ONE 3(3): e1736.
 
   Bose, I. and Karmakar, R. (2003) ‘Simple models of plant learning and memory’, Physica Scripta T106: 9-12.
 
   Brugger, P., Macas, E. and Ihlemann, J. (2002) ‘Do sperm cells remember?’, Behav Brain Res 136(1): 325-8.
 
   Chakravarthy, S. V. and Ghosh, J. (1997) ‘On Hebbian-like adaptation in heart muscle: a proposal for ‘cardiac memory”, Biol Cybern 76(3): 207-15.
 
   Corning, W. C. (1967) ‘Regeneration and retention of acquired information’, NASA.
 
   Corning, W. C. and Riccio, D. (1970) The planarian controversy, (ed. W. Byrne): New York: Academic Press.
 
   Costello, B., Adamatzky, A., Jahan, I. and Zhang, L. A. (2011) ‘Towards constructing one-bit binary adder in excitable chemical medium’, Chemical Physics 381(1-3): 88-99.
 
   Eisenstein, E. M. (1975) Aneural organisms in neurobiology, New York: Plenum Press.
 
   Gentile, L., Cebria, F. and Bartscherer, K. (2011) ‘The planarian flatworm: an in vivo model for stem cell biology and nervous system regeneration’, Dis Model Mech 4(1): 12-9.
 
   Hicks, C., Sorocco, D. and Levin, M. (2006) ‘Automated analysis of behavior: A computer-controlled system for drug screening and the investigation of learning’, J Neurobiol 66(9): 977-90.
 
   Holley, J., Jahan, I., Costello Bde, L., Bull, L. and Adamatzky, A. (2011) ‘Logical and arithmetic circuits in Belousov-Zhabotinsky encapsulated disks’, Phys Rev E Stat Nonlin Soft Matter Phys 84(5 Pt 2): 056110.
 
   Inoue, J. (2008) ‘A simple Hopfield-like cellular network model of plant intelligence’, Prog Brain Res 168: 169-74.
 
   Levin, M. (2011) ‘The wisdom of the body: future techniques and approaches to morphogenetic fields in regenerative medicine, developmental biology and cancer’, Regenerative medicine 6(6): 667-73.
 
   Levin, M. (2012) ‘Molecular bioelectricity in developmental biology: new tools and recent discoveries: control of cell behavior and pattern formation by transmembrane potential gradients’, Bioessays 34(3): 205-17.
 
   Lobo, D., Beane, W. S. and Levin, M. (2012) ‘Modeling planarian regeneration: a primer for reverse-engineering the worm’, PLoS Comput Biol 8(4): e1002481.
 
   McCaig, C. D., Rajnicek, A. M., Song, B. and Zhao, M. (2005) ‘Controlling cell behavior electrically: current views and future potential’, Physiol Rev 85(3): 943-78.
 
   McConnell, J. V. (1965) A Manual of Psychological Experimentation on Planarians. Ann Arbor, Michigan: The Worm Runner’s Digest.
 
   McConnell, J. V. (1966) ‘Comparative physiology: learning in invertebrates’, Annual Review of Physiology 28: 107-36.
 
   McConnell, J. V., Jacobson, A. L. and Kimble, D. P. (1959) ‘The effects of regeneration upon retention of a conditioned response in the planarian’, Journal of Comparative Physiology and Psychology 52: 1-5.
 
   Rilling, M. (1996) ‘The mystery of the vanished citations: James McConnell’s forgotten 1960s quest for planarian learning, a biochemical engram, and celebrity (vol 51, pg 589, 1996)’, American Psychologist 51(10): 1039-1039.
 
   Sheiman, I. M. and Tiras, K. L. (1996) Memory and morphogenesis in planaria and beetle. in C. I. Abramson Z. P. Shuranova and Y. M. Burmistrov (eds.) Russian contributions to invertebrate behavior. Westport, CT: Praeger.
 
   Shomrat, T. and Levin, M. (2013) ‘An automated training paradigm reveals long-term memory in planaria and its persistence through head regeneration’, J Exp Biol.
 
   Smalheiser, N. R., Manev, H. and Costa, E. (2001) ‘RNAi and brain function: was McConnell on the right track?’, Trends Neurosci 24(4): 216-8.
 
   Travis, G. D. L. (1981) ‘Aspects of the Social Construction of Learning in Planarian Worms’, Social Studies of Science 11: 11-32.
 
   Tseng, A. and Levin, M. (2013) ‘Cracking the bioelectric code: Probing endogenous ionic controls of pattern formation’, Communicative & Integrative Biology 6(1): 1-8.
 
   Volkov, A. G., Carrell, H., Adesina, T., Markin, V. S. and Jovanov, E. (2008) ‘Plant electrical memory’, Plant Signal Behav 3(7): 490-2.
 
   Wu, C. L., Shih, M. F., Lai, J. S., Yang, H. T., Turner, G. C., Chen, L. and Chiang, A. S. (2011) ‘Heterotypic Gap Junctions between Two Neurons in the Drosophila Brain Are Critical for Memory’, Curr Biol.
 

Michael Levin,
Tufts University
www.drmichaellevin.org/

Tal Shomrat,
Hebrew University

(5 votes)

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Here are the highlights form the current issue of Development:

Nf2 regulates neural progenitor proliferation

Mutation of neurofibromatosis 2 (NF2) results in nervous system tumours. Molecularly, Nf2 has diverse functions, regulating cell-cell junction formation and various signalling pathways, including the Hippo-Yap pathway. However, the roles of Nf2 in the nervous system, and how its loss promotes tumorigenesis, are poorly understood. Here (p. 3323), Xinwei Cao and co-workers analyse the consequences of Nf2 deletion in the dorsal telencephalon. Although the mutant mice are viable, they display significant brain malformations associated with neural progenitor cell (NPC) hyperproliferation. To determine how Nf2 limits NPC expansion, the authors performed a microarray analysis and found many known targets of the transcriptional coactivator Yap upregulated upon Nf2 deletion, suggesting that Nf2 may inhibit Yap activity. Consistent with this, protein levels and nuclear localization of Yap and its paralog Taz are increased in Nf2 mutants. Moreover, Yap deletion rescues the Nf2 mutant phenotype – demonstrating the functional importance of this regulation. These data uncover a key role for Nf2 and Yap/Taz in regulating NPC proliferation in the developing brain.

Staying in sync through development

During embryogenesis, transcriptional regulation must be coordinated with growth and cell division, so that genes are turned on or off in the right cells at the right time. Arjun Raj and colleagues now investigate the coupling of gene expression and cell division in C. elegans (p. 3385). They find that global retardation of development by temperature change or gene mutation slows down the cell cycle, and this is accompanied by a similar delay in expression of particular developmental genes – so the synchrony between cell cycle and gene expression is retained. These findings suggest that transcription might be directly cell cycle dependent. However, mutations that cause cell cycle delays in specific lineages uncouple cell division and transcription, arguing against the onset of transcription being tied to a particular division cycle. Conversely, it is known that cell division in C. elegans embryos proceeds independently of zygotic transcription. Together, these data demonstrate that cell proliferation and gene expression are well synchronised, but raise the key question of how this synchrony is achieved.

How cilia know which way to point

Cells lining the lumen of various organs, such as the lung airway and the female reproductive tract, are multiciliated, and all the cilia are oriented in the same direction to generate flow. But how is cilia orientation coordinated within cells and across tissues? Chris Kintner and colleagues use the epithelial cells of Xenopus embryos as a model to study multicilate cell differentiation. On p. 3468, they identify a new regulator of cilia polarisation, the coiled-coil protein bbof1. Bbof1 is expressed in multicilate cells and localises to the axoneme and the basal body – the structure that determines cilia orientation. Upon bbof1 depletion, motile cilia still form, but are unable to generate significant flow because their orientation is disturbed. Notably, bbof1 is not required for the initial phase of cilia polarisation, but rather for the later refinement step, and for stabilising the alignment. Although the mechanism by which bbof1 acts remains unclear, this work identifies a key factor regulating cilia orientation and function.

Go with the flow: circulating BMP promotes endothelial quiescence

Blood flow through the developing vasculature regulates vessel formation – both via the distribution of endocrine factors, and via mechanical force-induced responses. Several signalling pathways are known to be involved in this process, including signalling via the TGFb receptor Alk1, whose activity promotes quiescence in newly formed arteries and whose expression is itself dependent upon blood flow. On p. 3403, Beth Roman and colleagues demonstrate that not only Alk1 expression but also its activity are dependent upon blood flow in developing zebrafish. They identify Bmp10 as the endogenous ligand for Alk1 in this context, and find that Bmp10 is exclusively expressed in the heart, and not in the vascular tissue. Through elegant experiments using embryos in which the heart has been stopped but alk1 expression restored, they show that Bmp10 injection can locally rescue Alk1 pathway activity and downstream transcriptional responses. Thus, their data suggest that blood flow is required to distribute cardiac-derived Bmp10 into the vasculature, where it activates Alk1 to promote quiescence in endothelial cells.

Histone methylation: not so dynamic after all

Polycomb group proteins are chromatin regulators with highly conserved functions. The Polycomb repressive complex 2 (PRC2) methylates H3K27 to stably silence target genes, including the HOX genes in Drosophila. More recently, Utx and Jmjd3 demethylases were found to reverse PRC2-mediated H3K27 methylation, and it has been suggested that a dynamic cycle of methylation and demethylation is required for appropriate regulation of gene expression. Now, Ömer Copur and Jürg Müller challenge this view (p. 3478), via the analysis of Drosophila Utx mutants. Lack of zygotic Utx function has no effect on Drosophila development, although mutant adults die shortly after hatching. Loss of both maternal and zygotic Utx, however, leads to larval death and to defects in HOX gene expression – in both the embryo and larval imaginal discs. Thus, it appears that Utx in Drosophila – and, by inference, H3K27 demethylation – is required only at early stages to set up the patterns of HOX expression; it is largely dispensable later in development, suggesting that H3K27 methylation may in fact be very stable.

An integral role for integrin β1 in the pancreas

Integrins mediate cell-matrix adhesion and are also capable of inducing intracellular signalling cascades to regulate cell proliferation, differentiation and other cell behaviours. In vitro, disruption of β1 integrin function has been shown to affect various aspects of pancreatic β-cell activity. On p. 3360, Vincenzo Cirulli and co-workers analyse the consequences of deleting β1 integrin in β-cells in vivo in mice. The mutant mice have smaller pancreatic islets that exhibit matrix adhesion defects when cultured in vitro. Notably, cell proliferation is severely impaired in the mutant β-cells, and the expression of cell cycle regulators is highly abnormal. However, these cells are able to differentiate properly and to express insulin, and are glucose responsive; in fact, they show increased levels of insulin and the mutant mice show no signs of diabetes. These results highlight differences between the ascribed functions of β1-integrin in vitro versus in vivo and define its key role in promoting proliferation during pancreatic islet development.

PLUS…

Adult neural stem cells: plastic or restricted neuronal fates?

Eduardo Sequerro and colleagues review studies of postnatal and adult neurogenesis, challenging the notion that fixed genetic programs restrict neuronal fate. They hypothesize that the adult brain maintains plastic neural stem cells that are capable of responding to changes in environmental cues and generating diverse neuronal types. See the Hypothesis article on p. 3303

Clustered protocadherins

Weisheng Chen and Tom Maniatis provide a concise overview of the molecular and cellular biology of clustered Pcdhs, highlighting how they generate single cell diversity in the vertebrate nervous system and how such diversity may be used in neural circuit assembly. See the Development at a Glance poster article on p. 3297

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Matrone G, Taylor JM, Wilson KS, Baily J, Love GD, Girkin JM, Mullins JJ, Tucker CS, Denvir MA. Laser-targeted ablation of the zebrafish embryonic ventricle: A novel model of cardiac injury and repair. Int J Cardiol. 2013 Jul 17. doi:pii: S0167-5273(13)01117-0. 10.1016/j.ijcard.2013.06.063. [Epub ahead of print] PubMed PMID: 23871347 (Open Access Article)

In a study published July 17, 2013 in the International Journal of Cardiology on line, researchers found that “laser-targeted injury of the zebrafish embryonic heart is a novel and reproducible model of cardiac injury and repair suitable for pharmacological and molecular studies.”

The scientific team, led by Dr. Martin Denvir, College of Medicine and Veterinary Medicine, University of Edinburgh, UK, undertook this study to learn more about how the embryonic zebrafish heart responds to injury as compared to the adult zebrafish heart, which demonstrates a remarkable capacity for regeneration.

At the “heart” of this study was the XYClone infrared laser with RED-i target, from Hamilton Thorne Inc.. The researchers produced targeted and highly localized injury to the embryonic heart by synchronizing the XYClone laser pulse with the cardiac cycle. By using custom software, they were able to apply the laser pulse only at a specific user-designated phase of the cardiac cycle, which allowed targeting of just the embryonic heart ventricle.

Zebrafish embryos 72 hpf (lower panel)were used for all experiments of laser injury. The laser pulse was delivered to the area of the ventricle indicated by the red dot (Panel A) and resulted in a clear burn-mark at the point of injury (Panel B), see also supplementary movie 1 (V – ventricle, BA – bulbus arteriosus, At – atrium). Position of the embryo is marked by compass lines (c-caudal, cr-cranial, d-dorsal, v-ventral)

Cardiac arrest and cessation of tail blood flow demonstrated the immediate injurious effects of the laser. In addition, cell death and apoptosis resulted in loss of cardiomyocytes. A significant decrease in heart function was observed, yet, by 24 hours post-lasering, complete recovery occurred. The study results showed, for the first time, that a proliferation of new cardiomyocytes drove the functional recovery of the lasered embryo heart ventricle. It also appeared that the laser injury itself stimulated the proliferative process.

In the discussion, the authors note many advantages to using the laser model, including the rate at which the individual zebrafish embryos may be processed, the reproducibility, the ease of testing pharmacological and genetic interventions, and the ability to create regional damage similar to that which occurs from ligation of the coronary artery in mammals.

Supplemental videos:

Movie 1: Laser pulse injury (without synchronisation) of the zebrafish embryonic heart ventricle at 72 h post-fertilization– A single laser pulse, using the XYClone Laser Ablator, to the ventricle of a zebrafish embryo (72 hpf) results in instantaneous cardiac injury associated with marked bradycardia and gradual recovery of cardiac rhythm over the next few minutes. A laser burn-mark is clearly seen in the wall of the ventricle. This is an example where there is a clear view of non-overlapped cardiac chambers.

MOVIE 2: Laser pulse injury using the synchronization software of the zebrafish embryonic heart ventricle at 72 h post-fertilization. In this example, atrium and ventricle are overlapped. Attempting to injure the ventricle with a non-synchronized laser system would result in damage to adjacent structures. Synchronizing the laser pulse with the cardiac cycle allows highly precise and targeted injury to the ventricle at end-diastole and consequently minimizes damage to surrounding structures.

Note: The author, Cindy Rodzen, is affiliated with Hamilton Thorne Inc.

(1 votes)

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Dranow DB, Tucker RP and Draper BW. Germ cells are required to maintain a stable sexual phenotype in adult zebrafish. Developmental Biology 376: 43-50.

Adult sex-reversal  –  the change of primary sex (gonadal sex) and secondary sex characteristics and  to another sex during adulthood, occurs in many fish species and is triggered by social or environmental conditions.    This is an extreme example of phenotypic plasticity – the ability of animal to change its form due to a cue from the environment.   Sex reversal requires considerable changes to both the reproductive system (testis or ovary) and changes to secondary sex characteristics (such as pigment, body shape).

One example of sex-reversal occurring in nature is observed in some species of goby fish.  These fish get around as a group of females (harem) with a single dominant male.  With the loss of the male from group, one of the adult female fish (usually the largest) undergoes sex reversal to become the male of the group .

The prized fish model for studying development and modeling of human disease is zebrafish.   Adult zebrafish can also undergo sex-reversal induced by certain environmental conditions such as water temperature or the presence of aromatase inhibitors.  Despite being used for decades as a developmental and genetic model, the primary sex determination mechanism is unknown.    Recent research indicates that the primitive germ cells have a key role.  The developing gonad is made up of somatic cells  that form either the supporting cells or hormone producing cells of the gonad and the primitive germs cells which form the future sperm or oocytes.  In zebrafish gonad development goes through a transient stage where – no matter what the ultimate sex will be – both future male and female junvenile fish gonads contain primary oocytes.   In the male these early oocytes die off through programmed cell death and  the gonad (and the final morphological phenotype) develops as a male.  In females, the primary oocytes are maintained and the gonads differentiate into ovaries and the female secondary characteristics develop. For more information.

In a new article by Dranow et al., they examined the role of oocytes in maintaining the female sex phenotype in zebrafish.   To determine if loss of oocytes from adult female zebrafish could cause sex-reversal – they used two methods:

One method was to make use of a mutant animal that undergoes germ cell loss later in adulthood – nanos 3 null mutants.  Nanos proteins are a evolutionary conserved proteins required for survival of germ cells thus mutation or deletion of nanos  results loss of germ cells and infertility.  Zebrafish nanos3 is required to maintain the germ line – therefore nanos3 mutants are initially fertile, but by 5 months old females are now infertile due to the loss of oocytes during maturation.

Dranow et al., examined the sex characteristics of nanos3 mutant adult fish at 2.5 and 5 months.  Initially in nanos3 homozygotes (ie. have no functional copy of the nanos3 gene) the females had typical female phenotypic characteristics (at 2.5 months).  However by 5 months their overall body shape and pigmentation was now similar to that of a adult male zebrafish.  Indicating that the later life loss of oocytes (due to lack of nanos3) in the females adult mutants, resulted in female to male sex-reversal.

The second method they used was a transgenic approach to induce germ cell loss after chemical treatment.   They used transgenics to produce a line of zebrafish that expresses an enzyme in specifically in germ cells which induces cells to undergo cell death when exposed to a certain drug, metronidazole (Mtz).    One week following Mtz treatment,  the loss of oocytes from transgenic gonads was already evident and by two months the females were showing increased yellow pigmentation and body shape changes – ie. they were now more similar to a male phenotype.    Gonad histology and detection of sex-specific markers confirmed female to male sex reversal had occurred in the adult fish.   They were even able get 4 sex-reversed fish to induce females to spawn and produce viable embryos.    This indicates that sex-reversal is complete – initially adult females, following the loss of oocytes, now become sperm producing males.  This may give some clues as to the mechanism of natural forms of adult sex-reversal in other fish species.

What about mammals?  Evidence using mouse models suggest that somatic sex needs to be maintained/reinforced throughout adulthood.  Loss of FOXL2 (a gene required for ovarian development) expression in the adult ovary results in reprogramming of ovarian cell types (eg grandulosa cell) into cells types typically found in the testis, expressing male marker genes such as Sox9.    Like-wise deletion a gene required for male development in adult testes, DMRT1, results in loss of Sertoli cells (testis cell type) and new expression of female marker genes (ie. genes that are normally only expressed in the ovary).

Key summary points and remaining questions (from this paper and previous publications in this area)

1. Gonadal sex is surprisingly labile; the gender phenotype needs to be reinforced throughout adulthood.
2. Oocytes are required for development of female zebrafish   (both gonadal and secondary sex characteristics) in juveniles and maintain the phenotype in the adults.
3. Somatic cells in the juvenile appear to be bipotenial – induced to produce Sertoli or grandulosa cells and can be reprogrammed in the adult.
4. How can these cells be reprogrammed in an adult? ie.  What are the molecular mechanisms underlying sex-reversal of specialized differentiated cell types?
5. The primary signal of sex determination in zebrafish still remains a mystery but clearly the germ-somatic cell communication plays an important role in determining sex.

(13 votes)

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Like me, when you were a kid many of you probably puckered your lips and placed your hands on the side of you neck to make believe that you were a fish. At the time, you were probably just being silly and didn’t realize how similar you actually are to a fish. The zebrafish is a common model organism used in research because it shares a remarkable amount of genetic information with humans. Because of this similarity, the zebrafish model system has been useful for identifying and characterizing the genetics behind many different human birth defects, including anomalies to the craniofacial skeleton. It is increasingly clear that the environment plays an important role in the genesis of birth defects. Because zebrafish are fertilized externally, this tiny fish is a useful model for understanding these gene/environment interactions.

Gene/environment interactions may be beneficial, such as when a mother eats a healthy diet, or detrimental, like when an expectant mother is using drugs or alcohol. It is not new knowledge that alcohol can have detrimental impacts on the growth of the fetus. The first manuscript identifying Fetal Alcohol Syndrome (FAS) was published 40 years ago and the phenotypes associated with FAS were noted even earlier in children of alcoholic mothers. However, the effect of alcohol on a developing embryo can be incredibly variable, and the broader term Fetal Alcohol Spectrum Disorders (FASD) covers these variable, ethanol-induced phenotypes. FASD patients can suffer from both neural and craniofacial defects, and although timing and volume of exposure can influence the variability of this disease, genetic predisposition most likely adds to this variability.

In our study (McCarthy et al, 2013), we sought to identify genes that protected against ethanol-induced defects and, thus, might contribute to this variability. When these protective genes are mutated, embryos should be more susceptible to the deleterious effects of ethanol. Therefore, we exposed embryos to a level of ethanol that did not disrupt development in normal wild-type embryos. We examined several of the zebrafish mutant lines that we house in our fish facility for susceptibility to ethanol-induced developmental defects. We found that one of these fish lines, a hypomorphic pdgfra allele, was strikingly susceptible to ethanol. In fact, most of the heterozygous embryos, which still have one good copy of pdgfra, also had craniofacial defects; under normal conditions these heterozygotes do not have defects. We were particularly struck with how different the ethanol-treated heterozygotes and mutants looked from the untreated mutants. The difference in phenotypes suggested that ethanol interacted synergistically with pdgfra.

While untreated pdgfra mutants have defects in migration of skeletal precursors, ethanol treatment causes an increase in apoptosis of craniofacial precursor cells in ethanol-treated pdgfra mutants and heterozygotes. This provided support that pdgfra and ethanol did indeed synergistically interact. Mechanistically, we show that mTOR signaling, downstream of pdgfra, was reduced in ethanol-treated pdgfra mutants. mTOR is important in cellular growth and survival, and has been implicated to interact with ethanol in other studies. L-leucine is a common dietary supplement that can elevate mTOR signaling. Elevating mTOR signaling with L-leucine in ethanol-treated mutant zebrafish partially rescues the ethanol-induced phenotype. But the question remained does this information, obtained from a fish, help us understand FASD in humans?

Luckily for us, we were able to collaborate with the Foroud lab to get at this question. In a human sample, they found concordance between facial phenotypes and PDGFRA genotype, or  more specifically single nucleotide polymorphisms, SNPs, associated with the PDGFRA gene. This sample included children who either were or were not exposed to alcohol during pregnancy and who underwent a series of craniofacial measurements. Dr Foroud and her colleagues found a significant association between a Pdgfra SNP and differences in craniofacial measurements in ethanol-exposed children, compared to their unexposed counterparts. Importantly, the SNP had no effect on facial phenotypes by itself. It was only when the SNP was present and there had been an alcohol exposure that the phenotype was altered. Thus, what began as an interesting phenotype caused by exposing a little fish to ethanol has lead to a human SNP that might help us understand FASD.

Whether other genes, such as other growth-factor genes, could also interact with ethanol is of ongoing interest in the lab. Working with the Foroud lab, we hope to sustain the collaboration we have formed and build a better understanding of the genetic predispositions to FASD. By using a small fish we can provide the mechanistic details of these gene-ethanol interactions. So while we may not look much like fish, they can certainly tell us a lot about ourselves.

McCarthy, N., Wetherill, L., Lovely, C.B., Swartz, M.E., Foroud, T.M., and Eberhart, J.K. Pdgfra protects against ethanol-induced craniofacial defects in a Zebrafish model of FASD. Development 2013 Aug; 140(15): 3254-65.

(5 votes)

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Perhaps, like me, you’ve been microinjecting Xenopus embryos for so long that you start seeing strange things – maybe that they’re waving at you.  But perhaps that’s not so crazy as it sounds.  In a letter to Nature this week (you can also view this talk), Jeremy Chang and James Ferrell Jr. from Stanford discuss the evidence that contraction waves form along the surface of fertilised eggs, suggesting a mechanism for coordination of spatial information at the very earliest points of embryonic development.

Xenopus laevis eggs are large – greater than 1 mm in diameter.  Therefore you might think that the cell cycle is slow in these large, unwieldy cells, requiring longer diffusion times for molecules to move around the cell to regulate cytokinesis and cell division.  However, as anyone who has worked with these frog embryos is well aware, the early cell divisions are incredibly rapid – the first cell cycle is 90 minutes at room temperature, followed by a dozen rapid synchronous cell divisions every 30 minutes.  How is this rapid division related to the molecules that control it?

The authors suggest a mechanism revolving around maintaining Cdk1 activity, using positive and negative feedback loops that generate a bistable environment – essentially, causing waves of Cdk1 activity to propagate through the cell.  They refer to these “trigger waves” – which are waves caused by areas of high local (mitotic) activity causing nearby regions to flip from interphase to mitotic activity and therefore propagating a wave that spreads outwards.  Subsequent activation of APC/C and thus cyclin degradation would then trigger waves of mitotic exit, and if cyclin synthesis follows, a cycle is generated.

The authors discuss and propose this model, then looked for experimental evidence to test the hypothesis in the well-established extract systems that Xenopus laevis eggs provide.  By adding sperm chromatin and fluorescent nuclear-localising protein as reporters of mitosis to frog egg extracts, they were able to observe propagation of waves – there are videos attached to the paper you should watch – showing movement of the extract from interphase, to mitosis and back again. They link this directly back to the activity of Cdk1 by manipulating the feedback mechanisms of Wee1 and Myt1.

They then looked at intact fertilized eggs.  If the centrosome, an area of high Cdc25 and therefore Cdk1 activity, initiates trigger waves they could be observed as waves of contraction on the surface of the embryo, moving from the pigmented animal hemisphere at the top to the vegetal bottom.  The paper gives images illustrating this. Cdk1 is a kinase, which adds phosphate groups to proteins to affect their activity, and it is thought the changes in pigment are caused by the fibres being modified as the waves of activity spread out.

Of course, this isn’t just a frog thing (it’s never just a frog thing) – frogs provide an extreme example of the scale of the problem but it’s likely that this is how cell division occurs in embryo development and all situations where cells are dividing.  It’s a nice example of Xenopus laevis providing an excellent model for biophysical studies and models of the propagation of signals in cell division.

(7 votes)

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By Leonardo Valdivia and Joaquín Letelier

Here is a report about sessions performed during the 8^th European Zebrafish meeting held in Barcelona. The post is divided in different topics to make it easy to read.  Of course we did not cover all sessions and tried to focus on talks related to developmental biology, but we have also included some interesting talks beyond the main topic of the Node. We hope you enjoy it! (as we did).

Advances in Imaging

Vikas Trivedi started the session using two-photon SPIM microscopy to image the developing heart. He showed that during its formation a variety of cells are present in the heart primordium, perform different behaviors and adopt diverse morphology; these cellular features are essential for proper development of this organ. Next, Andrea Bassi showed a new technology for blood flow observation based on optical projection tomography (OPT). By rotating the samples in 360˚ a 3D reconstruction of the vasculature is obtained, which is then used to measure speed and direction of blood cells with no need of fluorescent labeling. Later on, Peter Eimon nicely showed a method for phenotyping embryos using whole animal tomography. With this analysis it is possible to do high resolution reconstruction of the embryos and measure different phenotypic features. Taking advantage of this technique (named hyperdimensional in vivo phenotyping) he showed that drugs that generate craniofacial defects produce similar phenotypic “signatures”. Finally, Gopi Shah showed a 4-lens SPIM system to obtain 2D projection images of a whole embryo in real-time, which allowed deep analysis of early zebrafish development.

Morphogenesis and Organogenesis

One of the recurrent topics in the meeting was morphogenesis. Given the nice and attractive conditions of our favourite model organism, attending these talks was a pleasure for the eyes and the mind.

Caren Norden kicked-off the session with an excellent talk about non-apical dividing progenitors in the developing retina and how the retinal architecture have a role on the emergence of this mode of division; she showed that retinal ganglion cells loss shift mitosis to basal locations, repositioning divisions in the developing retina. Keeping on eye formation, Toshiaki Mochisuki next examined the development of the lens. He explored into the cell dynamic within this structure and how cells change their position over time: cell divisions are the driving force for the cell movement observed during lens development and cell contacts influence cell division orientation and differentiation.

After dealing with the eye, the topic moved into heart development. In this part, Emily Noël talked about heart looping; by mutant analysis and explant culture, she proposed a pathway for amplifying a tissue intrinsic looping mechanism. Later on, Nadia Mercader showed beautiful in vivo imaging of heart beating and how this movement controls morphogenesis of the epicardium. She also showed amazing optical tweezing experiments, which allowed her to move single cells in the pericardial cavity. We really enjoyed it.

Esteban Hoijman introduced the next change in focus with a nice in vivo analysis of the inner ear formation. By using a breathtaking live imaging approach he nicely described the hollowing of this structure (lumen expansion), and showed is mediated by an actomyosin mesh and hydrostatic pressure. In the next talk, Nikoalus Obholzer provided a framework for system-level studies of the ear formation creating a 4-dimensional cell-based atlas of the ear. Such a good contribution will be helpful for testing hypothesis about ear formation.

Live imaging kept us amazed in the last two talks. Rachel Verdon presented a detailed view of pronephros development and glomerular filtration, providing a good model for kidney organogenesis. Finally, physics met the early embryo and Amayra Hernandez-Vega showed a dissection of the cellular mechanics driving epiboly, based in hydrodynamics as gastrulation goes on.

The talks provided a very good overview on several systems and a good taste about how the morphogenetic processes can be analyzed from different points of view and using different tools.

Gene Regulation and Genomics

This session started with an encouraging talk by Kerstin Howe from Sanger Institute about new improvements in the zebrafish reference genome that will result in a new version (GRCz10) in early 2014. Next, Todd Townsend introduced a novel method to get gene expression profiles from different tissues during development. His group developed an elegant system (BLRP-Rpl-BirA technology) to capture entire polysomes from specific cells with high affinity. With those samples they can determine which genes are active in confined cells during any developmental stage. Later on, Gustavo Gómez performed ChIPseq and RNAseq analysis to dissect the downstream targets of Etsrp/Etv2, a transcription factor essential for induction of vascular lineages. This work will help unravel new pathways involved in the development of vasculature. Finally, Miler Lee showed how maternal factors are cleared and zygotic genes activated during the maternal to zygotic transition. Using a sequencing approach on wild type and morphant embryos, he is getting deeper in the mechanisms that govern this essential process early in development.

New Technologies for Gene Manipulation

During the last years an explosive growth of new technologies has made fish an outstanding model for gene manipulation. This session provided a good update of some new tricks and resources that all the researchers in our field should know.

In the first talk Peggy Jungke introduced a productive gene trap screen for driving the expression of inducible Cre recombinase in different tissues of the embryo. These new zebrafish lines will allow the researchers to use conditional alleles or lineage-tracing approaches (for example the recently published zebrabow!). You can search for driver lines in http://crezoo.crt-dresden.de/crezoo/.

Carole Gauron was next, who introduced new optogenetic tools to manipulate cellular parameters in single cells or restricted tissues. She was able to induce and report apoptosis with this approach, opening new opportunities for regenerative studies.

It is well known that one of the most popular advantages of the zebrafish is transgenesis and Christian Mosimann introduced a new method to do it. He adapted a system used for single insertions of DNA fragments in predefined locus in fly, a technology that was not available for fish. This opens the possibility for inserting your favourite fragment of DNA in a simple, reliable and rapid manner. It will be helpful to increase the number and position of these landing sites in different fish lines to perform transgenesis in a highly controlled fashion. We look forward to hear more about it.

The next two talks aimed to show and compare current efforts for generating knock-out fish models using different approaches. Raman Sood compared the efficiency of ZFN (Zinc Finger Nucleases) and TALENs (Transcription Activator Like Effector Nucleases), giving useful parameters to keep in mind, especially for labs that are currently starting (or thinking) to work with these powerful custom designed nucleases. The next talk was given by Gaurav Varshney who introduced a new method for mapping insertional elements in the zebrafish genome, from a collection of mutagenised fish via this approach. This work is framed into the ambitious project for mutating all coding element in the zebrafish genome. Gaurav updated the efforts for the insertional mutants and encourage the audience to visit http://research.nhgri.nih.gov/ZInC/ for searching mutants for particular genes. The database is frequently updated, so keep an eye on that.

Finally, Koichi Kawakami (the father of the well known tol2 approach in zebrafish) reported the efforts for a large-scale Gal4 gene trap screening. He showed quite a few examples of specific pattern for those lines, providing reliable driver tools for manipulating UAS driven transgenes. All the lines that he showed (and many others) are available in http://kawakami.lab.nig.ac.jp/ztrap/. Importantly, he encouraged the audience to visit his lab in Japan for screening fish and find new drivers. His group can provide travel grants, so contact Koichi if you are interested!

Brain and Neural Crest Development

In this plenary session one of the most interesting talks (only because we like eye development!) was by Henrik Boije, who showed elegant data relevant to the formation of the retina. Using knockdown and transplantation techniques, he is dissecting how intrinsic and extrinsic factors can alter cell fate during retinal maturation. Next, Myriam Roussigne introduced the role of fgf signaling in the left-right asymmetry of the brain. She based her studies on the parapineal organ, a small group of cells migrating collectively from the midline to the left side of the developing brain. She showed fgf signaling activation restricted to few parapineal cells on the left side could be a key step for migration.

Finally, Angela Nieto showed how newly generated neurons maintain cell-cycle factors in a silenced state, as re-enter to this process leads to neuronal death. She placed scratch2 as a key regulator: knocking down this gene induces postmitotic neurons to the re-enter the cell cycle. Normal expression of scratch2 gene maintains high p57 (cell cycle inhibitor) by downregulation of miR-25.

Stem Cells and Regeneration

Fish has great potential for providing clues about tissue regeneration, something that is missed (or nearly) in mammals. Therefore a lot of attention is paid to understand how we can improve this process taking lessons from our favourite model. In this session the focus were stem cells (which are obviously linked to the regenerative process) and how they are controlled and organised in different context in zebrafish.

In the first talk, Michell Reimer nicely took advantage of combined approaches in fish. He showed that dopamine controls development and regeneration of motor neurons; this idea came up after a drug screen performed in his lab, encouraging the multidisciplinary approaches for finding completely new data. Alessandro Brombin then showed how cells in the brain contribute to the optic tectum and torus semicircularis. By using 4-dimensional time-lapse imaging, he tracked single cells all over the way. Given the recently published zebrabow approach, we can anticipate that similar strategies will be now possible in multiple colours including also cell shape in the equation.

Many of us use finclip for genotyping fish lines and we know empirically that after a few time the missing tissue grows back; fin regeneration is a well-established (and simple) model for tissue regeneration, but importantly it can be also used as a model for vertebrate limb regeneration. Mate Varga explored into the cellular mechanisms controlling this process: he showed that autophagy is increased during caudal fin regeneration in adults and this event is required for carrying out this task. In the same line of research, Rita Mateus explored into how the regenerated fin tissue knows how much to grow; she involved the Hippo pathway and mechanical interaction between the cells as critical players. Both talks were complementary and generated interesting questions.

David Stachura introduced a change from the regeneration topic, performing clonal analysis of hematopoietic stem cells and progenitors (which is a nice model for studying developmental restriction). He presented a successful assay to isolate and enrich hematopoietic progenitors for studying differentiation. In the future, using different and new transgenic lines could nicely complement it.

The next two talks investigated aspects of stem cell biology in the zebrafish retina. Vincent Tropepe talked about a mutant with defects in the ciliary marginal zone, a region in the postembryonic retina that contains stem cells allowing life-long growth of the fish eye. Mutants had an enlarged stem cell niche with decreased differentiation, meaning that the process is blocked in the transition. In the zebrafish eye there is a second stem cell population that is more active during regeneration: the Müller glia. Using a protocol for killing photoreceptors in the adult retinae, David Hyde talked about signals that make Müller glia to re-enter in the cell cycle for contributing to regenerate lost cells. He showed a multistep process in which dying photoreceptors secrete factors that are received by Müller glia, which then also start to produce and secrete them for amplifying the signal. This results in enhanced proliferation that helps to repopulate lost photoreceptor cells.

Finally, Laure Bally-Cuif showed data from an emergent model for neural stem cell biology: the adult pallium. She showed how notch3 dependent signalling gates cell cycle entry and limits neural stem cell amplification, proposing this pathway as an essential node of control.

All the talks were fantastic and covered different aspects from a broad point of view, which were carefully chosen by the organizers.

Cancer

Scientist interested in cancer use different techniques to study the growth, development and migration of tumour cells, and analysis in living organisms is imperative. In recent years, zebrafish has become an attractive model for studying this disease.

In this special session we learned what is currently possible to do with our favourite model organism. The speakers showed a combination of transgenic lines, genetic and molecule screens, and cell biology.

In the first talk Thomas Look emphasised that neuroblastoma is the most common extra cranial solid tumour in children. His group developed different transgenic lines as accurate models for inducing these tumours, which will be ideal for elucidating the cellular mechanisms of the synergy between oncoproteins. The next talk was by Marina Mione who explored post-transcriptional gene regulation by micro-RNAs during cancer transformation, a process by which a normal cell begins behaving like a tumour cell. She focused on melanoma and also generated transgenic lines displaying highly invasive cancer.

One big bottleneck for treatment is when cancer cells become resistant to therapies. Shelly Sorrells addressed this issue analysing mutants for genes involved in the resistant to ionizing radiation. Next, Paul Essers presented data about a zebrafish mutant model for the von Hippe-Lindau tumor suppressor gene and how it is required for the DNA damage response pathway.

Finding new molecules to block metastasis is always a challenge in the fight against cancer and Viviana Gallardo presented efforts towards this end. She showed a rapid in vivo screening for collective and single cell migration inhibitors, using the lateral line primordium (collective migration) and immune cell system (single migration) as they share molecular signatures and migratory behaviour with tumour cells. Some identified compounds showed a strong specific inhibitory effect (including some natural extracts); those results are very promising for finding new pharmacological treatments.

The last talk was by Yi Feng. She introduced another transgenic tumour model and showed astonishing movies of in vivo interaction between leucocytes and tumour cells, with immune cells surrounding and “biting” malignant cells. Amazing.

A big round of applause end up the session and we probably got more questions than answers, which is a good sign of research going forward.

We have to say that the whole meeting was very good organized and all worked perfect. Moreover, key lectures by Sydney Brenner and Denis Duboule were fantastic, showing us how far biology can go.

Now Barcelona is gone, but we look forward to Oslo 2015!

Our best fishes!

(14 votes)

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The field of modern genetics owes a lot to the pioneering work of Thomas Hunt Morgan on the little fruit fly. A great part of his work was conducted in a small, busy fly room at Columbia University, famous not only for the great science that was done there but also for its democratic social environment. At least 5 people who worked with Morgan or one of his students went on to win Nobel Prizes, and Morgan himself was awarded the Nobel prize in 1933 for his work on the role of chromosomes in heredity.

A new exhibition starting today in New York allows you to visit a reconstructed version of the 1920s fly room- including hundreds of milk bottles with fruit flies, vintage microscopes and original documents from the scientists including index cards with mutant fly drawings. There will also be projections of reenactments of events that happened there and macroscopic images of fruit flies. The exhibition is accompanied by a lecture series focusing on the importance of the discoveries in Morgan’s fly room in today’s research, taking place in the reconstructed room itself.

So whether you are a fly pusher yourself, or just an enthusiastic of the history of science, and you happen to be in New York, why not pop down to Pioneer Works in Brooklyn and visit the exhibition? If you are not lucky enough to have a chance to visit the exhibition, do not despair- the reconstructed fly room has also been used as film set! The feature film ‘The fly room’ focuses on the relationship between one of Morgan’s students, Calvin Bridges, and his 10 year old daughter, and is scheduled to hit cinemas early next year. So look out for it!

The Fly Room exhibition will be showing at Pioneer Works in Brooklyn, New York from the 24th of July to the 20th of August.

You can find out more about the exhibition and the lecture series here and you can read an article in The Scientist about the feature film or visit the film’s website.

(4 votes)
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Set against a beautiful backdrop of baroque architecture and cobbled streets in Murcia, Spain, the 6^th Zebrafish Disease Models workshop (ZDM6) kicked off with an awe-inspiring keynote talk on developing therapeutics using zebrafish, given by Leonard Zon. Regaling the audience with the stories behind two approaches that have shown concrete clinical promise since their discovery in the Zon lab – leflunomide-based treatment of melanoma and prostaglandin E2 in transplantation therapy – Leonard provided optimism to this community of researchers seeking to take fish studies from tank to bedside. Moreover, those who had travelled directly from the 8^th European Zebrafish meeting in Barcelona, where Leonard had closed the ‘Disease Models’ session, were delighted to hear about previously undisclosed, unpublished work with the potential to translate into novel therapies.

The first part of the meeting focused on haematopoiesis; the overriding themes were the identification of factors involved in the specification and maintenance of haematopoietic stem cells, and the dissection of signalling pathways that specify the critical timing of these events. Several speakers, including David Stachura and Trista North, emphasised that perturbations of haematopoiesis are associated with a variety of human diseases, so efforts to characterise the genetically similar haematopoietic system in zebrafish are important from a clinical, as well as a developmental, point of view.

On a more pragmatic note, Nathan Lawson began a discussion that persisted throughout the workshop on technologies for genetic manipulation of zebrafish. His lab has established and characterised a number of knockout mutants, providing useful models for understanding vascular biology and cardiovascular disease. However, using the analogy ‘the good, the bad and the ugly’, Nathan emphasised that reverse genetics approaches aren’t always successful for the elucidation of gene function, and the frequently observed phenotypic discrepancy between morphants and knockout mutants can lead to inconclusive findings. Many groups are now turning to genome editing technologies such as TALENs and the CRISPR/Cas system to generate new and improved fish models of disease.

Cancer was the major topic of the second part of the meeting. The zebrafish has proven to be an ideal model organism for studying tumour initiation and progression, as highlighted by Liz Patton, who gave an overview of her group’s pioneering work using zebrafish models of malignant melanocyte development. Melanoma incidence continues to rise, and the rate of relapse after therapy is discouragingly high. The identification of cellular factors that can induce melanoma regression is crucial for guiding new therapeutic approaches, as exemplified by the Patton group’s recent discovery of the role of MITF in melanoma onset and survival. As a cancer that can be robustly and reproducibly modelled in zebrafish, several zebrafish-based mechanistic insights into melanoma were presented in subsequent talks. By contrast, Manfred Schartl’s talk focused on the small aquarium fish medaka, which his group has exploited to examine the importance of miRNAs (and other non-coding RNAs) in melanoma pathology. In the Schartl lab, transgenic medaka that present with different types of melanoma in different genetic backgrounds have been generated, providing a unique platform for studying the pathological characteristics underlying various forms of the disease.

Another example of an good model for cancer was described by A. Thomas Look, whose lab recently developed a robust zebrafish model of neuroblastoma, an aggressive cancer of the peripheral sympathetic nervous system that accounts for 10% of all childhood cancer deaths. Using this model, Thomas showed that anaplastic lymphoma kinase (ALK) works in cooperation with the MYCN oncogene to drive neuroblastoma. The group is now using the model to examine synergy between other oncogenes and tumour suppressors in neuroblastoma, and also to determine the functional relevance of polymorphisms implicated by GWAS and sequencing studies. Given that neuroblastoma is clonal in nature, coding mutations are just the tip of the iceberg, stressed Thomas.

Multiple groups have generated zebrafish xenotransplantation models to study cancer. David Langenau, who had prepared two different talks and let the audience vote on which they’d prefer to hear, described how high-throughput transplantation methods in zebrafish can be used to track single cells and investigate cell heterogeneity in cancer. Jason Berman later gave a talk on the use of human tumour xenograft zebrafish models to identify drug targets and test the efficacy of anti-cancer therapies, bolstered by proof-of-principle studies of leukaemia. Claire Lewis’s success in using zebrafish xenotransplantation models to shed light on macrophage regulation of tumour relapse (after therapy) led her to describe herself as a ‘zebrafish convert’, having predominantly worked on mice until recently. Despite recapitulating the tumour microenvironment in a way that in vitro models cannot do, and being a more cost-effective and rapid system than mice xenografts, a discussion that followed several talks on this topic highlighted that further studies are needed to quantify the predictive capacity of zebrafish xenotransplantation models and firmly launch the approach into the pharmacological arena.

On the final day of the meeting, with the audience looking noticeably bleary-eyed after a night of fine-dining and flamenco dancing that lasted until the early hours, a whirlwind of impressive data were presented on infection and immunity. Annemarie Meijer talked about innate immunological defence mechanisms that, she emphasised, are relevant to all of the diseases discussed at the meeting. Recently, Annemarie and colleagues performed a functional analysis of a zebrafish line with a truncated version of Myd88, a key component in Toll-like receptor signalling. Using this tool, the group showed that the immune response to Salmonella and Mycobacterium infections differs at the level of gene expression, and provided evidence that Myd88 signalling has an important protective role in the early stages of tuberculosis. Talks by Astrid van der Sar and Herman Spaink further demonstrated the utility of zebrafish for modelling tuberculosis, while Serge Mostowy and Robert Wheeler presented data relevant to infection by Shigella flexneri or Candida albicans, respectively. Clearly, the range of infectious diseases that can be modelled using zebrafish is diverse and, frequently, these studies provide insight into immune pathways that are also relevant to complex diseases such as autoimmune disease and cancer.

All in all, the combination of an excellent scientific programme and a succession of lively networking events created yet another successful zebrafish disease models workshop. Attendees seemed to unanimously agree that the small size of the meeting and targeted sessions are a winning formula that they hope to emulate again next year, perhaps close in time and location to the 2014 GSA Zebrafish Genetics meeting in Madison, WI. James Amatruda, professor in paediatric oncology research at UT Southwestern Medical Center, and Jill de Jong from the Chicago Center for Childhood Cancer and Blood Diseases will take the baton from Maria L. Cayuela (University Hospital Virgen de la Arrixaca), and Jorge Galindo-Villegas and Victoriano Mulero (University of Murcia) as  organisers of the next meeting. If their enthusiasm for the task matches their zest for flamenco dancing, we are certainly in for a treat! Regardless of the location and format, DMM hopes to support the workshop again in subsequent years.

Paraminder Dhillon, DMM Scientific Editor

Selected DMM articles discussed at the workshop:

Research
Monique Anchelin, Francisca Alcaraz-Pérez, Carlos M. Martínez, Manuel Bernabé-García, Victoriano Mulero, and María L. Cayuela
Premature aging in telomerase-deficient zebrafish
Dis. Model. Mech. 2013, doi:10.1242/dmm.011635

Resource
Michiel van der Vaart, Joost J. van Soest, Herman P. Spaink, and Annemarie H. Meijer
Functional analysis of a zebrafish myd88 mutant identifies key transcriptional components of the innate immune system
Dis. Model. Mech. 2013 6:841-854, doi:10.1242/dmm.010843

Research
Remi L. Gratacap, John F. Rawls, and Robert T. Wheeler
Mucosal candidiasis elicits NF-κB activation, proinflammatory gene expression and localized neutrophilia in zebrafish
Dis. Model. Mech. 2013 dmm.012039; AOP

Read more DMM zebrafish-focused articles here:
http://dmm.biologists.org/site/collections/zebrafish.xhtml

(5 votes)

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The Wellcome Trust – Medical Research Council Stem Cell Institute draws together outstanding researchers from 25 stem cell laboratories in Cambridge to form a world-leading centre for stem cell biology and medicine. Scientists in the Institute collaborate to generate new knowledge and understanding of the biology of stem cells and provide the foundation for new medical treatments.

Principal Technician

Salary: £33,230 – £44,607pa

To assist the Institute Administrator in the day-to-day running of the Institute the key aspects of the role are: Leadership, Communication and Problem Solving.

The role holder will be responsible for building maintenance and security, implementation Health and Safety procedures, and organisation and supervision of the Institute’s technical and cleaning staff.  Experience in managing building projects and refurbishments is desirable.

You should be able to demonstrate experience in recruitment, supervision and performance management and will have excellent written and oral communication skills.  You will be responsible for procurement and purchasing of high value goods and services and must have a good understanding of financial accounting processes. Practical experience of computerised accounting packages and familiarity with University Financial System is desirable but training can be provided.

Educated to degree level or equivalent in a biological science or related subject, you will have previous management experience in a senior post in a science-related area in either higher education or industry setting as well as excellent motivational and interpersonal skills. Extensive experience in a biomedical environment including a period of hands on research is essential.

The building is multi-occupancy building and you will act as the Safety Officer for the Stem Cell Institute and the Cambridge Systems Biology Centre and will liaise with the Department of Chemical Engineering and Biotechnology in all matters relating to building maintenance.

To apply, please visit our vacancies webpage:

http://www.stemcells.cam.ac.uk/careers-study/vacancies/

Informal enquiries are also welcome via email: cscrjobs@cscr.cam.ac.uk

Applications must be submitted by 17:00 on the closing date of 8^th September 2013.

Interviews will be held week commencing 23^rd September 2013. If you have not been invited for interview by Tuesday 17^th September 2013, you have not been successful on this occasion.

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