This Spotlight article was written by Stefan Schulte-Merker and Didier Y. R. Stainier, and was first published in Development.

Morpholino oligomers have been used widely and for many years in the zebrafish community to transiently knock down the function of target genes. It has often been difficult, however, to reliably discriminate between specific and non-specific effects, and thus generally accepted guidelines to control for morpholino side effects do not exist. In light of recent methodologies to generate mutant lines in virtually any zebrafish gene, we discuss these different approaches with a specific focus on how the first description of a loss-of-function phenotype in zebrafish should be accomplished.

Initially, the genetic analysis of zebrafish development and physiology was dominated by mutants identified in small- and large-scale forward genetic screens (Chakrabarti et al., 1983; Driever et al., 1996; Haffter et al., 1996).Whereas forward genetics was instrumental in establishing zebrafish as an additional vertebrate model system, progress was hampered by the fact that there was no reliable technology to carry out reverse genetics in this model.TILLING (a reverse genetics approach based on high-throughput sequencing of ENU-mutagenized fish)was introduced only in 2002 (Wienholds et al., 2002) and requires considerable up-front investment in logistics and infrastructure. The introduction of morpholinos (MOs) in frogs (Heasman et al., 2000) and zebrafish (Nasevicius and Ekker, 2000) as an antisense reagent to transiently knock down gene function was therefore greeted with considerable excitement, as it appeared to fill a real void in the toolbox. Since its inception, countless studies using this technology have been published, including some using MOs to knock down maternally deposited transcripts to circumvent the generation of maternal-zygotic mutants, and others using caged MOs, which allow for inducible release of these antisense reagents (Shestopalov et al., 2012).

The MO antisense technology is based on nucleic acid bases that are linked tomorpholine rings and a non-charged phosphorodiamidate backbone. The rationale for this design was that MOs would not bind electrostatically to protein, hence causing less toxicity, while at the same time being resistant to nucleases (Summerton, 2007). MOs are injected into early zebrafish embryos using standard techniques. Commonly, they are ∼25-mers designed to be an exact antisense match against the region surrounding the first translated ATG (to block translation) or against a splice donor or acceptor site (to interfere with precursor mRNA splicing). It quickly became apparent that some MOs could work extremely well, and there are many MO phenotypes that efficiently mimic mutant phenotypes without any noticeable side effects. However, it has also become clear that MOs can lead to artifacts and that for many MOs the phenotypes caused by specific binding to the intended target RNA are difficult to separate from those caused by the non-specific binding to unintended targets (Eisen and Smith, 2008). In fact, a simple calculation suggests that binding to targets other than the intended precursor or mature mRNA is likely. A zebrafish embryo contains ∼500 ng of RNA, 2-5% of which is translatable (25 ng) (A. Giraldez, personal communication; see also Davidson, 1986). Assuming that at any given time there are more than 104 different mRNA species present in a cell (Davidson, 1986), and that those transcripts are equally represented among the 25 ng of mRNA, only 2.5 pg of a specific mRNA species is available for targeting. Injections typically deliver ∼1 ng of MO, often more. Assuming further that the target mRNA has an average length of 1.25 kb, whereas the MO is a 25-mer, this equates to a 2×104-fold molar excess of MO versus target mRNA. It is therefore most likely that this vast excess of MO will bind other RNA or other macromolecules. This situation would not be such a serious problem if there were reliable ways to distinguish specific from nonspecific effects. However, this is not the case, and one can at best only show that MOs affect the target sequence; non-specific effects cannot easily be identified, even when using mRNA rescues (see Del Giacco et al., 2010; Tao et al., 2011). The literature now contains several examples in which developmental delay, defects in organ asymmetry and pericardial edema (among many other ‘phenotypes’) are attributed to knocking down a specific gene, but in which subsequent generation of a mutation in that gene revealed a very different phenotype, and often no phenotype at all. Recent examples include mutations in sox18, nr2f1a and prox1a/b, all genes that had been reported to show morphant phenotypes within the lymphatic vasculature, whereas the mutant alleles do not (van Impel et al., 2014).

In several cases it has been possible to circumvent some of the non-specific phenotypes by suppressing p53 activity (Robu et al., 2007), which can reduce the ectopic cell death caused by nonspecific MO effects; however, this approach has its own caveats as it effectively generates a phenotype not on a wild-type, but on a p53-deficient, background. Applying such drastic corrective measures to allow a phenotypic analysis raises a number of questions that cannot be easily addressed.

Two surprisingly efficient alternatives for reverse genetics have been recently implemented in zebrafish (Chang et al. 2013; Huang et al., 2011; Sander et al., 2011; Hwang et al., 2013; Zu et al., 2013) and other organisms (Beumer et al., 2008; Tesson et al., 2011; Yang et al., 2013). TALE nucleases and the Crispr/Cas9 system are very efficient at generating mutations. As both techniques have been
reviewed extensively (Auer and Del Bene, 2014), we will restrict the discussion here to comparing the principles of the MO and TALEN/ Crispr approaches.

First, it should be noted that – like MOs – the implementation of these new technologies can be carried out in virtually any lab. Whereas TILLING really only makes sense for those willing to analyze large numbers of samples and genes, TALENs and Crispr do not require a substantial investment and, once established, can be used to generate targeting constructs within 1 (Crispr) to 2 (TALEN) weeks.

Second, as TALENs and Crisprs affect genomic DNA, rather than RNA transcripts, their molecular effect can be determined at the single embryo level (which is more difficult with MOs) to obtain a clear phenotype/genotype correlation. Of course, such an approach requires caution, as these nuclease-injected embryos are most likely to be mosaic for the resulting genomic lesions. Furthermore, TALEN and Crispr constructs can sometimes be efficient enough to generate loss-of-function situations in the actual injected embryos (Dahlem et al., 2012), and so, in a minority of cases, can be used almost like an MO; injection, scoring for phenotypes and confirming that the nuclease works efficiently can be performed within a few days.

Third, the published evidence, although currently limited, suggests that the side effects of these nucleases are often negligible (Hruscha et al., 2013), even though both TALE and Crispr-Cas nucleases can bind and cleave off-target loci (Reyon et al., 2012; Fu et al., 2014). When additional mutations are introduced, they can usually be segregated away from the mutation of interest by one or two outcrosses (as with mutations identified in ENU mutagenesis screens). This specificity is of course a tremendous advantage, and very different from MOs: an MO that binds non-specifically will most likely do so in every injected embryo. Lastly, it is relatively easy to generate multiple mutant alleles in one gene (e.g. by using TALEN pairs that affect different regions of the targeted gene), thus further reducing the chance of being misled by off-target mutations.

Hence, it seems fair to say that within the last year or so, the landscape of reverse genetics in zebrafish has changed, and it has changed for the better. Anyone can now, within a few weeks, generate reagents that can be used for reverse genetic experiments that appear to be of superior reliability and that are less burdened with side effects compared with MOs. Does that mean that we should do away with MOs altogether? Not necessarily: as we pointed out in the first paragraph, there are many MOs that are very useful and that appear to work specifically. We know they work specifically because we can compare them with a mutant phenotype. We would argue that this is the criterion that should be used in most cases: if one can show that an MO phenotype is an exact replicate of a mutant phenotype, then use of this MO is certainly acceptable and can save valuable time; for example, for injection into transgenic lines or for generating ‘double mutants’. However, the description of a phenotype that is provided for the first time and that is based solely on MOs without the ability to compare with a genetic mutant, should in the future be viewed very critically. In most cases, there are better alternatives in the form of nuclease based targeted approaches and there is no good reason not to use them.

References
Auer, T. O. and Del Bene, F. (2014). CRISPR/Cas9 and TALEN-mediated knock-in approaches in zebrafish. Methods pii: S1046-2023(14)00129-7 (in press).

Beumer, K. J., Trautman, J. K., Bozas, A., Liu, J.-L., Rutter, J., Gall, J. G. and Carroll, D. (2008). Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases. Proc. Natl. Acad. Sci. USA 105, 19821-19826.

Chakrabarti, S., Streisinger, G., Singer, F. and Walker, C. (1983). Frequency of gamma-ray induced specific locus and recessive lethal mutations in mature germ cells of the zebrafish, Brachydanio rerio. Genetics 103, 109-123.

Chang, N., Sun, C., Gao, L., Zhu, D., Xu, X., Zhu, X., Xiong, J.-W. and Xi, J. J. (2013). Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Res. 23, 465-472.

Dahlem, T. J., Hoshijima, K., Jurynec, M. J., Gunther, D., Starker, C. G., Locke, A. S., Weis, A. M., Voytas, D. F. and Grunwald, D. J. (2012).  Simple methods for generating and detecting locus-specific mutations induced with TALENs in the zebrafish genome. PLoS Genet. 8, e1002861. Davidson, E. (1986). Gene Activity in Early Development. Waltham: Academic Press.

Del Giacco, L., Pistocchi, A. and Ghilardi, A. (2010). prox1b Activity is essential in zebrafish lymphangiogenesis. PLoS ONE 5, e13170.

Driever, W., Solnica-Krezel, L., Schier, A. F., Neuhauss, S. C., Malicki, J., Stemple, D. L., Stainier, D. Y., Zwartkruis, F., Abdelilah, S., Rangini, Z. et al. (1996). A genetic screen for mutations affecting embryogenesis in zebrafish. Development 123, 37-46.

Eisen, J. S. and Smith, J. C. (2008). Controlling morpholino experiments: don’t stop making antisense. Development 135, 1735-1743.

Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M. and Joung, J. K. (2014). Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279-284.

Haffter, P., Granato, M., Brand, M., Mullins, M. C., Hammerschmidt, M., Kane, D. A., Odenthal, J., van Eeden, F. J., Jiang, Y. J., Heisenberg, C. P. et al. (1996).

The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 123, 1-36.

Heasman, J., Kofron, M. and Wylie, C. (2000). Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach. Dev. Biol. 222, 124-134.

Hruscha, A., Krawitz, P., Rechenberg, A., Heinrich, V., Hecht, J., Haass, C. and Schmid, B. (2013). Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish. Development 140, 4982-4987.

Huang, P., Xiao, A., Zhou, M., Zhu, Z., Lin, S. and Zhang, B. (2011). Heritable gene targeting in zebrafish using customized TALENs. Nat. Biotechnol. 29, 699-700.

Hwang, W. Y., Fu, Y., Reyon, D., Maeder, M. L., Tsai, S. Q., Sander, J. D., Peterson, R. T., Yeh, J.-R. J. and Joung, J. K. (2013). Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol. 31, 227-229.

Nasevicius, A. and Ekker, S. C. (2000). Effective targeted gene ‘knockdown’ in zebrafish. Nat. Genet. 26, 216-220.

Reyon, D., Tsai, S. Q., Khayter, C., Foden, J. A., Sander, J. D. and Joung, J. K. (2012). FLASH assembly of TALENs for high-throughput genome editing. Nat. Biotechnol. 30, 460-465.

Robu, M. E., Larson, J. D., Nasevicius, A., Beiraghi, S., Brenner, C., Farber, S. A. and Ekker, S. C. (2007). p53 activation by knockdown technologies. PLoS Genet. 3, e78.

Sander, J. D., Cade, L., Khayter, C., Reyon, D., Peterson, R. T., Joung, J. K. and Yeh, J.-R. J. (2011). Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat. Biotechnol. 29, 697-698.

Shestopalov, I. A., Pitt, C. L. and Chen, J. K. (2012). Spatiotemporal resolution of the Ntla transcriptome in axial mesoderm development. Nat. Chem. Biol. 8, 270-276.

Summerton, J. E. (2007). Morpholino, siRNA, and S-DNA compared: impact of structure and mechanism of action on off-target effects and sequence specificity. Curr. Top. Med. Chem. 7, 651-660.

Tao, S., Witte, M., Bryson-Richardson, R. J., Currie, P. D., Hogan, B. M. and Schulte-Merker, S. (2011). Zebrafish prox1b mutants develop a lymphatic vasculature, and prox1b does not specifically mark lymphatic endothelial cells. PLoS ONE 6, e28934.

Tesson, L., Usal, C., Ménoret, S., Leung, E., Niles, B. J., Remy, S., Santiago, Y., Vincent, A. I., Meng, X., Zhang, L. et al. (2011). Knockout rats generated by embryo microinjection of TALENs. Nat. Biotechnol. 29, 695-696.

van Impel, A., Zhao, Z., Hermkens, D. M. A., Roukens, M. G., Fischer, J. C., Peterson-Maduro, J., Duckers, H., Ober, E. A., Ingham, P. W. and Schulte- Merker, S. (2014). Divergence of zebrafish and mouse lymphatic cell fate specification pathways. Development 141, 1228-1238.

Wienholds, E., Schulte-Merker, S., Walderich, B. and Plasterk, R. H. A. (2002). Target-selected inactivation of the zebrafish rag1 gene. Science 297, 99-102.

Yang, H., Wang, H., Shivalila, C. S., Cheng, A. W., Shi, L. and Jaenisch, R. (2013). One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154, 1370-1379.

Zu, Y., Tong, X.,Wang, Z., Liu, D., Pan, R., Li, Z., Hu, Y., Luo, Z., Huang, P., Wu, Q. et al. (2013). TALEN-mediated precise genome modification by homologous recombination in zebrafish. Nat. Methods 10, 329-331

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Here are the highlights from the current issue of Development:

PCP signalling is dispensable for neural crest migration

The neural crest (NC) is a transient and migratory population of cells that gives rise to a variety of cell types. During development, NC cells delaminate from the neural tube in a process that is closely coordinated with the process of neural tube closure, and studies have shown that signalling via the planar cell polarity (PCP) pathway is essential for both of these processes in Xenopus and zebrafish. However, it is unclear if PCP signalling is required for NC migration in mammals. Here, Andrew Copp and colleagues address this issue (p. 3153). They show that NC specification, migration and tissue colonisation are not perturbed in mice that lack the function of the core PCP protein Vangl2. Furthermore, they demonstrate that Vangl1 does not compensate for the loss of Vangl2, as Vangl1/Vangl2 double-mutant mice also exhibit normal NC migration. The NC-specific ablation of Vangl2 activity also has no effect on NC migration. Finally, the researchers demonstrate that the migratory properties of NC cells from wild-type and Vangl2 mutant neural tube explants cultured in vitro are indistinguishable. Together, these findings confirm that, in contrast to its essential role in neural tube closure, PCP signalling is not essential for NC migration. Importantly, these findings also suggest that PCP mutations are unlikely to be the cause of NC-related birth defects in humans.

A helping Hand2 for heart development and regeneration

The production of cardiomyocytes is required both for embryonic heart formation and for cardiac regeneration following injury. The transcription factor Hand2 has been implicated in cardiomyocyte formation, but now, Deborah Yelon and colleagues demonstrate that Hand2 can in fact drive cardiomyocyte production in zebrafish (p.3112). They show that the overexpression of hand2 in early zebrafish embryos enhances the proliferation of cardiac progenitors within the second heart field, leading to increased numbers of cardiomyocytes and hence an increase in heart size. This cardiac enlargement also results from an increase in cardiomyocyte specification within the first heart field. Furthermore, they report, these effects require the phosphorylation-independent dimerization of Hand2 but not its direct binding to DNA. The researchers further investigate the role of Hand2 during regeneration and demonstrate that, in line with its role during development, hand2 overexpression can boost cardiomyocyte production following injury. These findings implicate the induction of hand2 expression as a key component of the cardiac regenerative response in zebrafish. Given that HAND2 has also been implicated in congenital heart disease (CHD) in humans, these findings also provide novel insights into the origins of CHD.

NR5A2: a central player in pancreas development

NR5A2 is an orphan nuclear hormone receptor that has diverse developmental and physiological functions. It is expressed in the inner cell mass of early mouse embryos and in the developing endoderm, but its role in organogenesis is unclear. Here, Ray MacDonald and co-workers reveal a crucial role for NR5A2 during pancreatic development in mice (p.3123). They first show that Nr5a2 is highly expressed in multipotent progenitor cells (MPCs), which give rise to endocrine, acinar and ductal cells, and in pre-MPCs, consistent with a role for NR5A2 in MPC formation. Indeed, pancreas-specific inactivation of Nr5a2 greatly diminishes the number of MPCs, and the development of all three lineages is affected. Subsequently, the researchers report, Nr5a2 expression is maintained in pre-acinar, acinar and ductal cells but is reduced in islet cells, suggesting that it regulates the development of the acinar lineage. In line with this, acinar morphogenesis was shown to be defective in an NR5A2-deficient pancreas. Furthermore, gene expression analyses indicate that NR5A2 can directly and indirectly modulate the expression of many genes involved in acinar differentiation and cell cycle control as well as in branching morphogenesis. These results demonstrate that NR5A2 controls multiple aspects of pancreas development and suggest that the experimental modulation of NR5A2 activity could be used to strategically direct the formation of pancreatic -cells in vitro.

Auxin keeps stomata in the dark

The development of stomata – the epidermal pores on plant leaves that regulate gas exchange – is tightly regulated by various environmental factors. Light, for example, promotes stomatal development; very few stomata are found on the epidermis of dark-grown seedlings. Here, on p.3165, Ute Hoecker and colleagues report that auxin, acting via Aux/IAA proteins, plays a key role in repressing stomatal development in dark-grown seedlings. The researchers show that aux/iaa mutants, which display auxin insensitivity, exhibit excessive stomata production specifically in dark-grown seedlings. This stomata-overproducing phenotype is also observed in mutants that are defective in auxin biosynthesis or perception, suggesting that auxin acts to repress stomatal production in the dark. They could further show that the excessive formation of stomata is caused by an increase in cell divisions within the stomatal lineage. Finally, the researchers use a combination of epistasis studies to elucidate a genetic network that integrates light and auxin signals in order to regulate stomatal development.

PLUS…

Out with the old, in with the new

Morpholino oligomers have been widely used by the zebrafish community for many years to generate loss-of-function phenotypes. Stefan Schulte-Merker and Didier Stainier reassess the usefulness of this methodology in light of recent developments in genome editing technologies. See the Spotlight on p.3103

Circadian clock-mediated control of stem cells

Recent research suggests that circadian clock mechanisms control more than just daily timekeeping. Here, Steven Brown discusses how such mechanisms can influence stem cell biology and hence tissue development, homeostasis and regeneration. See the Review on p.3105

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Science teachers usually say that science progresses by challenging old dogmas. In the stem cell field, there is a dogma saying that some blood stem cells in the bone marrow stay quiescent (do not divide) for long periods of time. This way, they avoid DNA damage and malignant mutations that could arise during DNA replication that happens during cell division. This ensures that some blood stem cells (also called haematopoietic stem cells, HSCs) keep their DNA intact, thus ensuring a healthy lifelong function.

However, a recent study published in Cell Stem Cell by Beerman and colleagues challenges this dogma by showing the quiescent HSCs accumulate DNA damage through the years, contrary to what was previously thought. Interestingly, these HSCs are able to repair this DNA damage when they have to divide.

In this picture, you can observe representative alkaline cornets of young HSCs on the left panel and old HSCs on the right panel. The cornet assay is a sensitive method to detect DNA damage in single cells. A single cell corresponds to a white circle and the more DNA damage there is the longer is the “tail”. You can observe that there are more cells with a “tail” in old HSCs on the right than in young HSCs on the left. When quantified, these differences are confirmed and demonstrate that there is more DNA damage in old HSCs than in young ones, showing that quiescence has not prevented DNA damage.

Altogether, this study refutes the dogma that HSCs are especially protected against DNA damage during aging. As a scientist, it is exciting and/or scary when dogmas are challenged: it is confusing, it raises new questions, but it ultimately makes science progress!

 Picture credit:

Beerman, I., Seita, J., Inlay, M. A., Weissman, I. L., & Rossi, D. J. (2014) Quiescent hematopoietic stem cells accumulate DNA damage during aging that is repaired upon entry into cell cycle. Cell Stem Cell, 15, (37-50). doi: 10.1016/j.stem.2014.04.016.

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Nowadays, the hardest thing in science is similar to what we experience in daily life, that is organization and choice. In a virtual plethora of techniques, methods and analyses, an aspiring researcher is faced with a flood of information, achievements and tools of the trade. Especially so with computers. But if it wasn’t for this ongoing torrent, some great synthetic approaches in studying biological variation would not emerge. Geometric morphometrics is an example of one such approach, as it greatly benefited from the ever increasing informational content all around biology, mathematics, statistics and computer science. If one is exposed to this multitudinous information, then organizational skills must take over natural curiosity, which is not very likely. Another important choice to be made regards the way creativity is shaped, the way it is displayed for others to see. This choice presents a balance between organization and chaos, a natural creativity border. In the blog Creative Morphometrics I choose some tools, R, python and octopress blogging platform, to try to tell one small part of the story about the wonderful world of biological shape analysis, its visualizations and the ways it connects to underlining phenomena, such as ontogeny.

For now, not much underlining and outward connections has been made in the blog, but its concept will be adapted over time (hopefully it is adaptable, at least in the design). Each post mostly deals with one particular idea, a problem in shape analysis and its proposed solution, along with results and R/python code. Of course, the emphasis, for now is put to techniques and tools (R or python packages) which can produce results that are both visually appealing and informative. Python-related posts are more basic (as my knowledge of python) and are there to “bring” geometric morphometrics to python, since there are no readily available packages for these analyses. R posts are the creative part (debatable creativity), with some not so common tasks in morphometrics, but also in data analysis in general.

One common point between quantification of morphological variation and its underlining developmental interactions, especially in focus within the concept of evo-devo, is modularity in the complex morphological structures. All morphometric analyses are underpinned by modularity, as it is one of the most important determinants of variability of both parts and the hole of adult morphology. The data I was working on during my PhD thesis research, mammalian crania (Figure below), exhibited constant and complex pattern of modularity across populations, and future posts in Creative morphometrics will surely include more aspects of modularity, as well as techniques that will enable the a-posteriori (purely analytical) determination of modules. Modularity represents the pattern of covariation between parts that share common developmental origin or form the same functional unit. Most informative papers about this key concept are “Evolution of covariance in the mammalian skull” by Hallgrimmson et al., “Morphological integration and developmental modularity” by Klingenberg and “The road to modularity” by Wagner et al. They bring closer the observed phenotypic variability and the developmental interactions that can both produce and maintain patterns of covariance in the complex morphological structures, enabling better understanding of the interactions between development, morphology and the environment.

Developmental modules of the chamois goat skull (orange – predominantly neural crest, blue – predominantly somitomeric origin of the cranial elements).

In conclusion to this post, the title can be repeated, as, with R, python and tremendous computational and analytic power available today our natural scientific curiosity can only sigh that there are so many tools and so little time. Researchers must make choices and only through communication with others will these choices flourish and bring forth the results.

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They are a mouthful, paper titles, sometimes. This is exactly the sort of title that would have made me ignore it in the days when I worked on the evolution of Hox genes. But I now find myself frequently justifying to people who work on evolution why the nervous system deserves attention, and of justifying to neuroscientists why the evolution of their systems is interesting (the latter is harder). Carving out a scientific niche is a very difficult and arduous task at the best of times and requires careful reflection, insight and planning. Above all though, it requires luck. What I have learned thus far about successful scientists though, is not that they make their own luck, as some would have you believe; they don’t. It is that when an interesting observation or finding rears its head, they follow it up. Even if it hadn’t already occurred to them. In fact, especially when it hadn’t already occurred to them. Intellectual flexibility is crucial. Nevertheless, it is apparently good to have a plan so this post is part of my effort to develop one. It is hardly Marx and Engels, but then, I don’t think they had EvoDevo in their day.

So, this post carries the same title as a recent paper that I published with an incredibly talented PhD student, Micha Hanzel, and my former supervisor, Richard Wingate at KCL. I have been invited to write a brief post about it on here by the boss of all things Nodal (ha! Sorry…), Cat Vicente. I normally balk at this kind of shameless self-promotion (sometimes), but I thought that I would indulge in it now for two reasons: to outline why it’s interesting, and to discuss what I mean by the word ‘interesting’. If I stray into the territory of ‘my work is cool because…’ you have my permission to slap me when you next see me.

Our work was on the evolution of development underlying the complex cerebellum found in amniotes. Actually not all amniotes, just birds and mammals. Essentially, they have massive numbers of neurons in the cerebellum and as such a massive cerebellum that is all stereotypically wrinkly in much the same way as the neocortex (the cool bit of the forebrain) of primates. All other groups of vertebrates don’t possess this foliated structure, which has been crucial to amniotes doing all sorts of cool things like manipulating things with their paws/hands, flying, and in primates, thinking. That is not to say that there aren’t a huge variety of interesting cerebellums around the rest of the vertebrate phylogeny – there are – but I was interested in how the amniote one got so impressive*.

Being comparative biologists, we characterised cerebellar development throughout the life cycle of the frog, which like I suspect the tetrapod ancestor had 300 million years ago, has a very simple structure with zero foliation. Now in amniotes, massive amplification of stem cells occurs weirdly on the basal (‘outside’) surface of the cerebellum, the complete opposite of where most progenitors live in the brain (on the inside, next to the ventricle). Given the lack of large numbers of neurons in the frog, we assumed that this external layer would be absent. Early in development it was, but to our surprise, we found the layer much later in development around metamorphosis. However, it was totally non-proliferative. In amniotes the transcription factor NeuroD1 normally acts to cause cell cycle exit and is turned on just as cells leave the external layer; in frog, it was expressed ‘prematurely’ in the external layer.

This led us to try to re-capitulate this condition experimentally in the chick (I had an interview recently where one of the academics said ‘’you chick people, you have it too easy!’’ But it is nice to have experimental manipulation so readily possible). ‘Premature’ NeuroD1 misexpression is indeed capable of causing cell cycle exit and we were able to show, using some funky (well, I think so) combinations of cis regulatory reporter constructs, that it down-regulates the gene Atonal1, which normally facilitates proliferation. Happy days: delaying expression of a single transcription factor enabled the evolution of a stem cell population that drove cerebellum evolution in amniotes.

Now, this is not going to change the world immediately in any predictable way, it won’t solve medulloblastoma (a devastating childhood cancer which develops from the external layer), and it certainly doesn’t address any of the strategic priorities that the grant proposal originally had to be measured against. But it is interesting. Not that it went into Nature or Science, and not that it will revolutionise the way lots of other people do science. It probably (sorry editors/future me) won’t have high ‘impact’. I mean, of course I hope it does, because I am part of this daft system where non-professionals judge my science by the effect it has on the gross domestic product of the UK or some other completely misguided and frankly idiotic metric assessment of an activity that is by its very nature not metrical. I do, though, think it is interesting. Because it explains, in some small way, something about some aspect of the natural world which was previously unexplained. Which is what science is meant to do, isn’t it?

To be clear then, I hope that my work reflects me. It is absolutely and determinedly not ‘cool’. I hate that word. It seems to have replaced ‘interesting’ in the scientific lexicon. It’s nearly as bad as ‘impact’. Why is it that as people, we grow up and stop being slavishly concerned (most of us) about whether other people think we are cool, or copy trends that we set, in our late teens or early 20s, and yet as scientists many of us chase exactly that folly and judge others by it? I don’t care (or rather, I shouldn’t) if no-one copies my experimental or philosophical approach in a way that leads to new insight into the process of root tip growth, the development of circuits implicated in autism, or the dynamics of arthropod leg diversity. They may do, and if so, I will care (and be happy!) about it then. But not now: science is the ultimate unpredictable pursuit. When people pretend otherwise and ‘sales pitch’ their work based on some defined future benefit, they are at best slavishly colluding in a system that is to the ultimate benefit of no one (which at present we all have to do), or at worst downright lying. This work on brain evolution is not ‘cool’ or ‘impactful’ (my all time most hated word), but it is interesting for its own sake and that is all anyone should care about.

*There is enthusiastic controversy surrounding the nature of the cerebellum in fish, and I have partially contributed to this, arguing that it possesses nothing like the same developmental complexity, but I could well be wrong, and so in the interests of civility am giving that topic a wide birth here.

 
Butts, T., Hanzel, M., & Wingate, R. (2014). Transit amplification in the amniote cerebellum evolved via a heterochronic shift in NeuroD1 expression Development, 141 (14), 2791-2795 DOI: 10.1242/dev.101758

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This obituary first appeared in Development. Also read other obituaries about Walter Gehring in Science, EMBO Journal and Developmental Cell.

Alex Schier looks back at the life and research of his graduate mentor and friend Walter Gehring.

Walter Gehring, one of the fathers of modern developmental biology, died on May 29th 2014 from the injuries sustained in a car accident. Gehring and his collaborators led the field, from experimental embryology to molecular genetics. On the way they discovered the homeobox, isolated master regulators of segment identity and organ formation, developed enhancer trapping and helped reveal the remarkable conservation of developmental control mechanisms in animals.

Gehring grew up in Switzerland and was trained as a classical zoologist, gaining his first research experience with radar studies of bird migration. As a graduate student with Ernst Hadorn, he studied transdetermination, the process by which, upon repeated transplantation, imaginal discs can change their identity from one segment to another (Gehring, 1966b). During that time, Gehring also found a gain-of-function allele of Antennapedia, which transformed the antennae of a fruit fly into legs (Gehring, 1966a). The realization that tissues can be transformed from one fate into another through transplantation or mutation defined Gehring’s future path and his quest to identify the molecular basis of tissue identity.

Gehring joined Yale University in 1965, first as a postdoctoral fellow in Alan Garen’s laboratory and then as an assistant and associate professor. He attempted to purify DNA-binding proteins that differed between imaginal discs but was initially unsuccessful in these efforts and retreated to more tractable projects (Chan and Gehring, 1971; Shearn et al., 1971). In 1972 he returned to Switzerland and became one of the founding members of the Biozentrum at the University of Basel. It was at the Biozentrum that he and his co-workers helped revolutionize the field of developmental biology over the next 25 years.

Gehring was one of the early disciples of the molecular cloning revolution. Following in the footsteps of his friend Dave Hogness (Bender et al., 1983; Glover et al., 1975), he and his team isolated some of the first Drosophila genes and then embarked on the cloning of Antennapedia (Artavanis-Tsakonas et al., 1977; Garber et al., 1983; also see Scott et al., 1983). This tour de force was rewarded with the monumental discovery of a DNA segment – the homeobox – that was conserved not only between Antennapedia and other Drosophila homeotic genes but also in other animals, including humans (McGinnis et al., 1984a,b,c; also see Scott and Weiner, 1984). Not unjustly, the homeobox has been called the Rosetta Stone of developmental biology: it was the blueprint for the isolation of developmental control genes in all animals and laid the molecular foundation for the field of evo-devo (Slack, 1984).

In quick succession, Gehring and his collaborators identified scores of homeobox genes, revealed their expression patterns, dissected their regulatory regions, and determined how the homeodomain binds to DNA in vitro and in vivo (Fjose et al., 1985; Hafen et al., 1984; Hiromi and Gehring, 1987; Hiromi et al., 1985; Kuroiwa et al., 1984; Mlodzik et al., 1985; Qian et al., 1989; Schier and Gehring, 1992). Remarkably, when Gehring and his co-workers ectopically expressed Antennapedia in transgenic flies, they induced the same antenna-to-leg transformation that he had observed 20 years earlier as a student (Schneuwly et al., 1987). This experiment was not only the culmination of 20 years of research but it also provided the proof-of-principle that tissues can be reprogrammed and redesigned in vivo, a quest that is ongoing in the field of regenerative medicine.

During that time, Gehring and his group also pioneered in situ hybridization and developed enhancer trapping and thus revealed some of the most stunning patterns in biology, ranging from gradients and stripes to fine-grained arrangements in the nervous system (Bellen et al., 1989; Hafen et al., 1984, 1983; Levine et al., 1983; Mlodzik et al., 1985; O’Kane and Gehring, 1987; Wilson et al., 1989; also see Akam, 1983). These iconic images were only surpassed by the eyes on the legs of Drosophila, generated by the misexpression of eyeless (Pax6) (Halder et al., 1995). Discovered serendipitously, eyeless was found to be both necessary and sufficient for eye development, and supported the concept of organ selector genes (Halder et al., 1995; Quiring et al., 1994). Even more importantly, the broad conservation of Pax6 in all animals and its expression in eye precursors overturned the idea that the eye had evolved independently dozens of times. Instead, Gehring postulated that the strikingly diverse eyes found in the animal kingdom derived from an ancestral eye (Gehring and Ikeo, 1999). Thus, the discoveries of the homeobox and Pax6 were not only thrilling scientifically, but they also highlighted philosophical and even ethical implications about the shared heritage of all animals.

Most recently, Gehring took up the challenge set forth by his hero Charles Darwin – how could “organs of extreme perfection and complication” evolve – and began to search “for the gradations through which an organ in any species has been perfected” (Darwin, 1859). Gehring postulated a symbiont theory for eye evolution, which he dubbed the ‘Russian Doll’ hypothesis (Gehring, 2014). He supported the idea that light sensitivity first arose in cyanobacteria, which were later engulfed by red algae as primary chloroplasts. In turn, the red algae were taken up by dinoflagellates as secondary chloroplasts and transformed into complex photoreceptive organelles. Finally, he imagined that through endosymbiotic gene transfer dinoflagellate genes were transferred to cnidarians. It now falls to the next generation of evolutionary developmental biologists to test Gehring’s Russian Doll hypothesis.

Today, we take many of the concepts and technologies developed by Gehring and his colleagues for granted. But these breakthroughs happened at a time when many developmental biologists were still following the credo of classical embryology and focused on cutting and pasting tissues between embryos. Indeed, Development only gained its current name in 1987; previously going by the title Journal of Embryology and Experimental Morphology. Gehring’s path reflected, predicted and guided this transformation in the field and this publication. Fittingly, he served as an editor for Development from 1990 to 1992 and on the editorial advisory board for many years, and published one of his first and one of his last papers in this journal (Gehring, 1966b; Prince et al., 2008).

Gehring received numerous awards, from the Kyoto Prize for Basic Science (2000) to the Grosses Bundesverdienstkreuz of Germany (2010). Like many great scientists, Gehring had his egocentricities (Gehring, 1998) and detractors (Gehring, 1999; McGinnis and Lawrence, 1999) but he had a magic touch in attracting highly motivated colleagues, who he enabled and encouraged to pursue risky, high-impact projects in an environment that functioned like an artist colony. A visitor to his laboratory in the late 1980s and early 1990s would have witnessed students transplanting imaginal discs, cloning developmental control genes or dissecting enhancers; postdocs studying the biochemistry of homeodomain-RNA and -DNA interactions, testing homologous recombination in flies or cloning enhancer-trapped genes; and technicians analyzing the heat shock response in desert ants. His influence on the community is reflected in the list of his former laboratory members, which includes Eric Wieschaus, Janni Nüsslein-Volhard, Paul Schedl, Spyros Artavanis-Tsakonas, David Ish-Horowicz, Ruth Steward, Renato Paro, Ernst Hafen, Mike Levine, Bill McGinnis, Atsushi Kuroiwa, Yash Hiromi, Marek Mlodzik, Cahir O’Kane, Henry Krause, Greg Gibson, Leslie Pick, Ylva Engstroem, Tony Percival-Smith, Shigeru Kondo, Ueli Grossniklaus, Markus Affolter, Hugo Bellen, Clive Wilson, Ken Cadigan, Georg Halder and Patrick Callaerts, to name
just a few.

Gehring was not only a visionary scientist and mentor but he was broadly engaged in research enterprise and education. He was an inspiring teacher of genetics, evolution, marine biology and developmental biology and co-author of the leading German textbook in zoology. He was a pioneer in training molecular biologists in Europe during the early days of the cloning revolution, and was a generous supporter of EMBO, serving as Secretary General (1996-2001) and helping lay the foundation for the ascendance of Europe in the field of molecular biology. Gehring and his colleagues, including Werner Arber, Gottfried Schatz, Max Burger and Eduard Kellenberger, made the Biozentrum one of the most exciting institutes in the world for modern biology. Together with his outstanding assistant Erika Wenger, he organized
many inspiring conferences in remarkable settings, and during the anti-immigration movement in Switzerland in the 1970s, he and his colleagues stood up for foreign scientists.

There was a special playfulness to Gehring. When he found the Antennapedia allele, he named it Nasobemia, inspired by Christian Morgenstern’s poem ‘Das Nasobem’, which describes a hopeful monster walking on its nose. And when he led his marine biology students through Banyuls-sur-Mer, a beautiful village in the south of France, he would profess about the secrets of wine and how to pick and peel figs and eat coquille St. Jacques. He would travel to remote places to observe and photograph his beloved birds and, more recently, butterflies. Gehring’s passion and knowledge spanned entire fields, from DNA-protein interactions to bird migration, and from French wine to eye evolution. We will miss one of the last renaissance men in biology.

References:

Akam, M. E. (1983). The location of Ultrabithorax transcripts in Drosophila tissue sections. EMBO J. 2, 2075-2084.

Artavanis-Tsakonas, S., Schedl, P., Tschudi, C., Pirrotta, V., Steward, R. and Gehring, W. J. (1977). The 5S genes of Drosophila melanogaster. Cell 12, 1057-1067.

Bellen, H. J., O’Kane, C. J., Wilson, C., Grossniklaus, U., Pearson, R. K. and Gehring, W. J. (1989). P-element-mediated enhancer detection: a versatile method to study development in Drosophila. Genes Dev. 3, 1288-1300.

Bender,W., Akam, M., Karch, F., Beachy, P. A., Peifer, M., Spierer, P., Lewis, E. B. and Hogness, D. S. (1983). Molecular genetics of the bithorax complex in Drosophila melanogaster. Science 221, 23-29.

Chan, L.-N. and Gehring, W. (1971). Determination of blastoderm cells in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 68, 2217-2221.

Darwin, C. R. (1859). On the Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races in the Struggle for Life. London: John Murray.

Fjose, A., McGinnis, W. J. and Gehring, W. J. (1985). Isolation of a homeo box containing gene from the engrailed region of Drosophila and the spatial distribution of its transcripts. Nature 313, 284-289.

Garber, R. L., Kuroiwa, A. and Gehring, W. J. (1983). Genomic and cDNA clones of the homeotic locus Antennapedia in Drosophila. EMBO J. 2, 2027-2036.

Gehring, W. (1966a). Bildung eines vollständigen Mittelbeins mit Sternopleura in der Antennenregion bei der Mutante Nasobemia (Ns) von Drosophila melanogaster. Arch. Klaus Stift. Vererb. Forsch. 41, 44-54.

Gehring,W. (1966b). Uebertragung und Aenderung der Determinationsqualitaeten in den Antennenscheiben-Kulturen von Drosophila melanogaster. J. Embryol. Exp. Morphol. 15, 77-111.

Gehring, W. J. (1998). Master Control Genes in Development and Evolution: The Homeobox Story. New Haven: Yale University Press.

Gehring, W. J. (1999). Lifting the lid on the homeobox discovery. Nature 399, 521-522.

Gehring, W. J. (2014). The evolution of vision. Wiley Interdiscip. Rev. Dev. Biol. 3, 1-40.

Gehring, W. J. and Ikeo, K. (1999). Pax 6: mastering eye morphogenesis and eye evolution. Trends Genet. 15, 371-377.

Glover, D. M., White, R. L., Finnegan, D. J. and Hogness, D. S. (1975). Characterization of six cloned DNAs from Drosophila melanogaster, including one that contains the genes for rRNA. Cell 5, 149-157.

Hafen, E., Levine, M., Garber, R. L. and Gehring,W. J. (1983). An improved in situ hybridization method for the detection of cellular RNAs in Drosophila tissue sections and its application for localizing transcripts of the homeotic Antennapedia gene complex. EMBO J. 2, 617-623.

Hafen, E., Kuroiwa, A. and Gehring,W. J. (1984). Spatial distribution of transcripts from the segmentation gene fushi tarazu during Drosophila embryonic development. Cell 37, 833-841.

Halder, G., Callaerts, P. and Gehring, W. J. (1995). Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. Science 267, 1788-1792.

Hiromi, Y. and Gehring, W. J. (1987). Regulation and function of the Drosophila segmentation gene fushi tarazu. Cell 50, 963-974.

Hiromi, Y., Kuroiwa, A. and Gehring, W. J. (1985). Control elements of the Drosophila segmentation gene fushi tarazu. Cell 43, 603-613.

Kuroiwa, A., Hafen, E. and Gehring, W. J. (1984). Cloning and transcriptional analysis of the segmentation gene fushi tarazu of Drosophila. Cell 37, 825-831.

Levine, M., Hafen, E., Garber, R. L. and Gehring,W. J. (1983). Spatial distribution of Antennapedia transcripts during Drosophila development. EMBO J. 2, 2037-2046.

McGinnis, W. and Lawrence, P. A. (1999). Historical transformations. Nature 398, 301-302.

McGinnis, W., Levine, M. S., Hafen, E., Kuroiwa, A. and Gehring, W. J. (1984a). A conserved DNA sequence in homoeotic genes of the Drosophila Antennapedia and bithorax complexes. Nature 308, 428-433.

McGinnis, W., Garber, R. L., Wirz, J., Kuroiwa, A. and Gehring, W. J. (1984b). A homologous protein-coding sequence in Drosophila homeotic genes and its conservation in other metazoans. Cell 37, 403-408.

McGinnis, W., Hart, C. P., Gehring, W. J. and Ruddle, F. H. (1984c). Molecular cloning and chromosome mapping of a mouse DNA sequence homologous to homeotic genes of Drosophila. Cell 38, 675-680.

Mlodzik, M., Fjose, A. and Gehring, W. J. (1985). Isolation of caudal, a Drosophila homeo box-containing gene with maternal expression, whose transcripts form a concentration gradient at the pre-blastoderm stage. EMBO J. 4, 2961-2969.

O’Kane, C. J. and Gehring, W. J. (1987). Detection in situ of genomic regulatory elements in Drosophila. Proc. Natl. Acad. Sci. USA 84, 9123-9127.

Prince, F., Katsuyama, T., Oshima, Y., Plaza, S., Resendez-Perez, D., Berry, M., Kurata, S. and Gehring, W. J. (2008). The YPWM motif links Antennapedia to the basal transcriptional machinery. Development 135, 1669-1679.

Qian, Y. Q., Billeter, M., Otting, G., Müller, M., Gehring, W. J. and Wüthrich, K. (1989). The structure of the Antennapedia homeodomain determined by NMR spectroscopy in solution: comparison with prokaryotic repressors. Cell 59, 573-580.

Quiring, R., Walldorf, U., Kloter, U. and Gehring, W. J. (1994). Homology of the eyeless gene of Drosophila to the Small eye gene in mice and Aniridia in humans. Science 265, 785-789.

Schier, A. F. and Gehring, W. J. (1992). Direct homeodomain-DNA interaction in the autoregulation of the fushi tarazu gene. Nature 356, 804-807.

Schneuwly, S., Klemenz, R. and Gehring, W. J. (1987). Redesigning the body plan of Drosophila by ectopic expression of the homoeotic gene Antennapedia. Nature 325, 816-818.

Scott, M. P. and Weiner, A. J. (1984). Structural relationships among genes that control development: sequence homology between the Antennapedia, Ultrabithorax, and fushi tarazu loci of Drosophila. Proc. Natl. Acad. Sci. USA 81, 4115-4119.

Scott, M. P., Weiner, A. J., Hazelrigg, T. I., Polisky, B. A., Pirrotta, V., Scalenghe, F. and Kaufman, T. C. (1983). The molecular organization of the Antennapedia locus of Drosophila. Cell 35, 763-776.

Shearn, A., Rice, T., Garen, A. and Gehring, W. (1971). Imaginal disc abnormalities in lethal mutants of Drosophila. Proc. Natl. Acad. Sci. USA 68, 2594-2598.

Slack, J. (1984). Developmental biology: a Rosetta stone for pattern formation in animals? Nature 310, 364-365.

Wilson, C., Pearson, R. K., Bellen, H. J., O’Kane, C. J., Grossniklaus, U. and Gehring, W. J. (1989). P-element-mediated enhancer detection: an efficient method for isolating and characterizing developmentally regulated genes in Drosophila. Genes Dev. 3, 1301-1313

(4 votes)

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Hello everyone.

My background is in systems engineering, and i have over forty years experience of building and trouble shooting mechanical/hydraulic systems. I have an interest in the evolution and function of biological systems, hence my involvement in this. I have no issues with the genetic basis of biology, but i am pretty sure that genes cannot produce structures that can defy the laws of physics! There are physical laws that any system has to take account of, and that can also be taken advantage of in the function of the system. In my opinion the structures ultimately created by genetics, have evolved to take advantage of such physical laws. The example described here can be demonstrated in modern humans, and has wider implications in gender related physiology.

There is a physical connection in the mammalian dermal system, that has not been factored into certain dermal changes. This involves the mammalian hair cycle. There are many references to this disintegration and re-enlargement of the hair follicle “pocket” within the dermal tissue. The ongoing mystery of this is how cycle by cycle, the resulting follicle can change size so changing the amount of hair produced?

What is not refered to in any of the literature, is the natural resistence of the dermal tissue to such an enlargement of a pocket within it? Two things cannot occupy the same space at the same time. A hollow pocket in particular, is going to be very sensitive to external pressure. I suggest once you consider this basic physical connection, the mystery begins to clear.

Given recognised dermal physiology, the implication is that the hair producing structure evolved as a re-cycling pocket to use the prevailing resistence of the surrounding tissue. This is the most simple way to integrate hair production with the primary temperature response in mammals. The variable resistence of dermal tissue effects the follicle size, through the normal spacial restrictions upon tissue growth described here:

http://www.pnas.org/content/early/2014/03/26/1323016111

There are other advantages in evolution for the pocket based structure of the hair producing unit in mammals. More details are given in my basic outline article here:

DERMAL EVOLUTION PDF

This includes examples in modern humans, of the principles involved in this dermal resistence factor. I suggest people try out the eyebrow test described for themselves. There is much that can be added to this basic outline article. This just intends to describes the basics, and my thoughts on the wider implications.

One important implication, is that a particular androgen significantly increases lymphatic drainage efficiency. This represents a gender difference that has not been previously considered, but would fit very well with the known gender differences in immune function. This could shed an important light upon female susceptibility to autoimmune disease, and i am trying to promote the testing of this question.

Regards.

Stephen Foote.

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Here are last month’s highlights!

Research and news:

– Jacqueline wrote about her recent paper in Development, investigating the developmental origin and evolution of turtle shell patterning.

– This month’s Stem Cell Beauty post focused on the role of two transcription factors in muscle development… and how the way such discoveries help us get closer to a therapeutic application is not unlike football.

– and the European  Molecular Biology Organisation (EMBO) celebrated 50 years this month, so we collated some of the Node posts in the last few years with a connection to EMBO.

 Videos:

– What happens when you mix comedy with developmental biology? The answer is the Devo Show, a comedy performance that took place at the Society for Developmental Biology meeting and which you can watch in full on the Node.

– and we posted the Waddington Medal lecture given by Phil Ingham at the recent Spring meeting of the British Society for Developmental Biology. It provides an interesting overview not only of his career but also of the Drosophila  and zebrafish fields.

  Also on the Node: 

– Simon wrote about Microscopes4Schools, an outreach project that aims to bring microscopy to the classroom.

– Henrique and Rodrigo showed that Brazil is not just about football and sun, by providing an overview of the past and present of developmental biology in this country.

– Thomas shared his thoughts on the recent Young Embryologists Meeting.

– And a second round of beautiful images from last year’s Woods Hole embryology course was up for voting. Congratulations to Brijesh Kumar– his zebrabow image will feature in the cover of a future issue of Development.

Happy reading!

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Every year the British Society for Developmental Biology (BSDB) awards the Waddington Medal, its highest accolade, at the society’s Spring meeting. This year the Waddington Medal was awarded to Prof Phil Ingham, a geneticist and developmental biologist well known for his contributions to the field: from his PhD work on Drosophila‘s trithorax, to the identification of the vertebrate hedgehog genes. Phil gave a fascinating talk at the meeting, not only providing an overview of his career but also interesting insights into the Drosophila and zebrafish fields.

We were  involved in recording this lecture, which is now available in full at The Company of Biologists YouTube channel and below. You can also find out more about Phil by reading our interview with him, which was posted here on the Node (and in Development) last month.

(1 votes)

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We can now announce the winner of this year’s 2nd round of images from the Woods Hole embryology course: the ‘zebrabow’ zebrafish!

The full results were as follow:

– Short-tailed fruit bat; 264 votes
– Butterfly ovariole: 90 votes
– Mouse embryo: 32 votes
– ‘zebrabow’ zebrafish: 272 votes

Many congratulations to Brijesh Kumar (Indian Institute of Technology, Kanpur), who took this image at last year course. The image shows a “zebrabow” zebrafish (Danio rerio) embryo, 2 days post-fertilization. 4-Hydroxytamoxifen (4-OHT) was administered at 24 hr post-fertilization, leading to ubiquitous expression of active Cre recombinase, and subsequent expression of GFP, RFP and CFP after recombination.  It was imaged on a Zeiss LSM 700 confocal.

The other great images in this round were taken by Mary Colasanto, University of Utah, and Sophia Tintori, University of North Carolina (short-tailed fruit bat); Ezgi Kunttas-Tatli, Carnegie Mellon University, and Duygu Ozpolat, University of Maryland (butterfly ovariole); and Georgina Stooke-Vaughan, University of Sheffield (mouse embryo).

The winning zebrabow image will feature in the cover of a coming issue of Development. Look out for another round of beautiful images soon!

(1 votes)

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