The other beauty of Fluorescent Reporter Gene Systems is its undeniable likeness to holiday colours. So then, will we eventually see Christmas trees with genes hooked up to GFP, YFP and RFP?

The purposeful beauty of the system is in our ability to view artificial fluorescence (and hence the genes they report) throughout development in living tissue. There’s no need to freeze tissues in lethal chemicals to obtain results. Furthermore, we can observe fluorescence from teeny organelles under the confocal microscope to whole plants under a UV lamp. (Fantastic, now how do you switch it off?)

(Image: Ethereal looking Arabidopsis seed with glowing red nuclei, Development Cover Oct. 2009. Read more about it in the original article by Fitz Gerald et al., 2009)

Since the advent of RNAi, they’re now being used as molecular sensors for the occurrence of gene silencing in vivo. Recently, RNAi signal transport in tissues was uncovered using GFP. Researchers constructed a small RNA directed against the GFP sequences. What they found was quite surprising. If they injected their small RNAs in one leaf of a tobacco plant, which is widely expressing GFP, over time the GFP diminished first in the veins and then in tissues. Eventually, the fluorescence was quenched (summarized in Voinnet 2002).

Here in Australia, Peter Waterhouse (a Grand-daddy of RNAi in plants) is working on making live action movies of RNAi in progress. One new aim in his lab is to produce the “Zebrafish” of plants…a chlorophyll-less Arabidopsis plants. Weird or genius?

(Image: Tobacco plant afflicted with a virus can cause an unusual green glow similar to GFP. The red is autofluorescence from chlorophyll. Viral transport can overlap with the transport of RNAi signals, which also serves as a plant defense mechanism against viruses & other foreign nucleic acids. Flikr CC, by Xmort)

While beautiful, it’s a not perfect system in plants for a variety of reasons. The main one is autofluorescence, or the bright red grinch that inadvertently contrasts with the merry green. A microscope tech described it as nothing short of a total nightmare. Plants naturally fluoresce because of the pigments they contain ~ chlorophyll. The huge heap of noise it makes can block the signal from the reporter

gene. However, it’s getting easier to get around this using a range of light filters. Some filters only allow a narrow wavelength of light to go through. Usually they’re specific to just the wavelength of the GFP/RFP/YFP signal, and this causes non-fluorescing tissue to appear black.

(Image: my own hideous encounter with autofluorescence :(, note that chlorophyll is absent in the pollen & petals).

At any rate, I won’t keep my fingers crossed for that fluorescent Christmas tree. But it could be potential electricity saver. Instead of purchasing a bunch of energy consuming twinkle lights, simply purchase a fluorescent bulb (doesn’t have to be UV) and switch it on in place of your regular house lights. And if you get tired of the fluorescence, spray or inject some RNAi on a branch.

On a side note, while looking for possible videos on reporter genes, I came across the following video on Youtube:

Growing Nerve Cells from Hair Follicle Stem Cells in Mice?

So…stem cells in hair follicles…highly related to nerve & brain stem cells? Exciting new trend..or weird?

Thanks for reading!

YFP in the Heart Stage embryo of  Arabidopsis thaliana (Gifford 2003)

References:
Gerald, J., Hui, P., & Berger, F. (2009). Polycomb group-dependent imprinting of the actin regulator AtFH5 regulates morphogenesis in Arabidopsis thaliana Development, 136 (20), 3399-3404 DOI: 10.1242/dev.036921

Gifford, M. (2003). The Arabidopsis ACR4 gene plays a role in cell layer organisation during ovule integument and sepal margin development Development, 130 (18), 4249-4258 DOI: 10.1242/dev.00634

(3 votes)

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(This interview by Kathryn Senior originally appeared in Development on December 21, 2010)

Ben Scheres is an expert in plant development. He has been investigating development in Arabidopsis at Utrecht University, The Netherlands, since 1990, where his group uses the root tip as an easily accessible supply of plant stem cells. Ben agreed to be interviewed by Development to talk about his interest in stem cells and the beauty of self-organisation in plants.

When did you first realise you were going to follow a science career?

When I was 14, we visited a fancy science museum called ‘Evoluon’ and I bumped into a very lucid explanation of how DNA ‘encoded life’. I still remember the big spiral staircase model and the impression it made on me. Even as a small kid I had often wondered how life works – and here was a model that seemed to make it possible to start to understand some of that!

What first made you interested in stem cell research in plants?

For my PhD, I chose a research project in the field of plant-microbe interactions. At that time, breakthrough papers in fly and worm development were appearing thick and fast and I was fascinated by them. I realised, then, that we were nowhere near being able to describe the development of plants in similar detail. I looked for a plant system that had the same clear cellular relationships that we see in C. elegans and would present similar genetic possibilities. It was also important for the system to have the developmental flexibility that characterises plants. Arabidopsis roots fitted the bill and I have been hooked on them ever since.

What is the most striking difference between animal and plant stem cells?

First of all, plants do not set apart a germline, so all stem cells are somatic. In animals, many somatic stem cells have quite a restricted potential, but this is not the case in plants. There are far fewer restrictions and stem cells can also be easily regenerated. Induced pluripotent stem cells in animals have created quite a stir but this is no big deal in plants. Of course, this raises lots of questions and we’d like to understand much better what determines this difference.

(more…)

(4 votes)

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Boning up on stem cell Igf2-P2 function

The insulin-like growth factor (IGF)/insulin signalling pathway regulates cell proliferation, differentiation, aging and life span. During embryonic development, transcription of the mouse and human Igf2 gene is tightly regulated by four alternative promoters whose specific roles are unclear. Now, Sylvie Nathalie Hardouin and colleagues reveal that the transcriptional activity of one of these promoters, Igf2-P2, regulates mesenchymal stem cell differentiation and osteogenesis in mice (see p. 203). The researchers show that Igf2-P2 loss-of-function mice, in which a lacZ-neo cassette replaces the P2-driven transcriptional unit of Igf2, have short, thin, poorly mineralised bones and exhibit altered bone remodelling. These abnormalities are associated with decreased numbers of embryonic mesenchymal chondroprogenitors, adult mesenchymal stem cells and osteoprogenitors. Together, these and other results support a model in which the transcriptional activity of the Igf2-P2 promoter regulates the fate of mesenchymal progenitors during bone development and adult bone remodelling, and regulates osteogenesis through its effects on both osteoprogenitors and their microenvironment.

X inactivation: from imprinted to random

In female mammals, one X chromosome is epigenetically silenced in adult cells by the process of X inactivation (Xi). However, in the pluripotent epiblast cells of the preimplantation mouse embryo, both X chromosomes are active and Xi of the paternal or maternal X occurs at random shortly after implantation (random Xi). By contrast, in very early mouse embryos (and in extra-embryonic lineages), the paternal X chromosome is selectively inactivated (imprinted Xi). So when exactly does the mode of Xi change from imprinted to random during development? On p. 197, Hitoshi Niwa and colleagues examine Xi during the differentiation of inner cell mass (ICM)-derived female mouse embryonic stem (ES) cells. The researchers use forced expression of Cdx2 and Gata6 to induce ES cell differentiation toward trophectoderm (TE) and primitive endoderm (PrE), respectively. They report that random Xi occurs in both TE and PrE cells and in the TE of cloned embryos derived from female ES cells, suggesting that all marks for imprinted Xi must be erased by the time the ICM forms.

Muscle building: a connective tissue workout

Muscle and its connective tissue are intimately linked during embryogenesis and adult life. Thus, interactions between these tissues might be crucial for their development. To date, the lack of molecular markers for connective tissue fibroblasts has hindered the study of these potentially important interactions, but now, on p. 371, Gabrielle Kardon and colleagues identify the transcription factor Tcf4 as a marker for connective tissue fibroblasts and reveal that connective tissue is an important regulator of myogenesis. By making Tcf4^GFPCre mice, which allow genetic manipulation of connective tissue fibroblasts, they show that these fibroblasts regulate both muscle fibre type and maturation. In addition, the researchers unexpectedly discover that low levels of Tcf4 in myogenic cells promote the overall maturation of muscle fibre type. These and other data identify novel extrinsic and intrinsic mechanisms that regulate myogenesis and show for the first time that connective tissue is a vital component of the niche that controls muscle development.

Mitochondrial pathway central to fly apoptosis

Apoptosis (programmed cell death) is essential for development and tissue maintenance in many organisms. In mammals and C. elegans, Bcl-2 family proteins facilitate apoptosis by regulating mitochondrial dynamics but do they play a similar role during apoptosis in Drosophila? According to Kimberly McCall and co-workers, the answer to this question is yes in the Drosophila ovary (see p. 327). During mid-oogenesis in flies, apoptosis is induced in some of the egg chambers in the ovary when nutrients are scarce. The researchers show that, during this event, the mitochondrial networks of ovarian nurse cells undergo extensive remodelling, cluster formation and cluster engulfment by somatic follicle cells. These mitochondrial dynamics, they report, are dependent on caspases, the Bcl-2 family, the mitochondrial fission and fusion machinery and the autophagic machinery. Furthermore, cell death in the ovary is defective in Bcl-2 family mutants. Thus, the researchers conclude, Bcl-2 family proteins do play a major role in controlling both mitochondrial dynamics and cell death in the Drosophila ovary.

Retinal cell fates largely left to chance

Classic experiments in invertebrates suggest that stereotypic patterns of cell division generate specific cell types during development, but the extent to which stereotypic lineages play a part in the developing vertebrate CNS is an open question. Now, Michel Cayouette and co-workers report that stochasticity plays a major role in cell fate decisions in the developing rat retina (see p. 227). In vivo cell-lineage tracing studies show that vertebrate retinal progenitor cells (RPCs) yield retinal clones of varying size and cellular composition. Whether this variability reflects distinct but reproducible lineages among many different RPCs or stochastic fate decisions within a population of more equivalent RPCs is unclear. To find out, the researchers use videomicroscopy to follow the lineages of rat RPCs cultured at clonal density. Their analysis of the reconstructed lineages indicates that fixed probabilities determine the decision of the RPCs to multiply or differentiate. Thus, stochasticity plays a major part in the development of the retina and possibly also of other parts of the vertebrate CNS.

Worming out organiser evolution

Axial organisers, embryonic regions that induce cell fate and establish body axes during development, have been identified in various metazoans but their evolutionary origins and conservation of function remain unclear. The presence of an axial organiser in annelids (ringed worms) has not previously been confirmed, but now Takashi Shimizu and colleagues provide direct evidence that, in Tubifex tubifex annelids, descendants of a single blastomere of 4-cell embryos can function as axial organisers (see p. 283). The first two cleavages of T. tubifex embryos generate four macromeres (A-D) that subsequently divide to generate micromeres. The researchers show that ablation of the D macromere descendants 2d and 4d can inhibit axial development. Co-transplantation of 2d and 4d into ectopic positions, they report, induces secondary axis formation in host embryos; in these axes, neurectoderm and mesoderm derive from the transplanted micromeres, whereas the endoderm derives from the induced host. These studies identify D quadrant micromeres as annelid axial organisers, informing future studies of axial organiser conservation and evolution.

Plus…

During nervous system development, axon branching allows elaborate synaptic connections to form. Recent advances, reviewed by Daniel Gibson and Le Ma, identify how various axon branching morphologies develop and the common principles that regulate them.
See the Review article on p. 183 of this issue.

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Some of the most popular posts on the Node have been those about career prospects for young scientists. The category pages for job ads and career posts are among the most visited parts of the site, but neither of them has had as many hits as the discussion titled “too many postdocs and PhD students?”

In the comments of that post, back in July, Greg Dressler wrote:

“I do think we need to get over the idea that nothing short of an academic career fulfills the ideal goal of our students and post-docs. Most of the folks I went to graduate school with are not in academics anymore, yet they have meaningful and successful careers.”

And James Briscoe added:

“More flexibility is what’s needed and the acknowledgment and encouragement of a diversity of career routes and development paths.”

To follow on these thoughts we’ll profile a range of alternative careers for developmental biologists on the Node. Over the next few months we’ll have posts up from several people who found a career away from the bench. All posts in this series will be tagged altcareers, so you can easily find them all on one page.

We have already approached a few people to ask them to share their story, but if you would like to add your own experience in finding work outside of academic research, feel free to register for the Node and add a post with the altcareers tag, or contact us to get a set of guiding questions if you’d like some help with writing.

Eventually, we’ll summarize all responses in a feature article.

I’ll kick off the series with my own story in a few days. Spoiler: I’ll complain about the phrase “alternative careers”, because for me it was never an alternative to begin with!

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With people in many countries preparing to take a few days off at the end of this month, and other countries starting their summer break, I’m sure many of you have had to deal with the stress of handling your experiments over the holidays. How do you explain to a tank of zebrafish or a flask of cells that you’re going to visit your family for a few days? Do you just not go at all? Is the whole lab leaving feeding instructions with that new postdoc who lives just across the street from the institute and comes in every day anyway?

Let us know via the poll, and leave a comment if you want to explain your answer.

(poll closed and archived)

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If anyone is up to listening to lectures in French, and it’s not so bad, really, there are a series of excellent lectures in developmental and stem cell biology available from the STEM-Pole, a federation of Paris-region laboratories interested in stem cells from a more fundamental or more therapeutic perspective. These took place during an annual meeting in mid-November in Paris, that was live webcast.

Last year’s webcast, I was sick in bed, but not only was I able to listen during the talks but I even asked a question of two of the speakers. Some of those talks are also still online.

The EasyCast Nomad hard-/software that was used for the recording is among the lightest and easiest for the end-user that I have experienced. You not only see and hear the speaker, but also their slides in real time, coordinated with their talk.

I was privileged to participate in a preserved-for-posterity teleconference (good thing I forgot my webcam that day!) with a number of developmental biology educators about the Next Generation Embryology project in Newcastle, England. Go check out the project – it’s quite fantastic, really, even if you are not a human embryologist. But the software, as flexible and amazing as it seems (great for teleconferencing, really), is a bit unwieldy, and I manage to crash it on a regular basis even on replay only.

Nature Network, for its annual Science Online conferences, has made ample use of webcasting software to mixed results. I attended the 2008 blogging conference, so couldn’t say how casting went. The 2009 one, was at 3 a.m. from my point of view on vacation in the U.S., and so I literally participated in my pajamas, using the suggested Second Life interface. It was a bit gimmicky but somewhat functional – again, the software was too unwieldy for easy interaction with speakers, and the avatar business a bit distracting though with still a lot of potential. There is quite the Second Life science community over there at Nature Network, for those who are interested, with virtual seminars at the Elucian Islands.

Anyone have other webcast experiences to share, either examples to follow or to avoid?

(1 votes)

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The backstory to our recent Developmental Biology paper “The secreted integrin ligand nephronectin is necessary for forelimb formation in Xenopus tropicalis”  includes scenes of several members of the Zimmerman lab peering at a tank of metamorphosing transgenic frogs, scratching their heads, and agreeing that some of them “looked funny” (the frogs, not the researchers) before realizing that while their hindlimbs had formed normally, their forelimbs were completely absent.

In winter of 2005, I had been working for some time in Lyle Zimmerman’s lab at the NIMR, London, bringing genetic analysis to the study of amphibian developmental biology. Xenopus tropicalis, unlike the closely related allo-tetraploid Xenopus laevis, is a true diploid, with one of the smallest tetrapod genomes, and is amenable to genetic analysis. By the end of 2005, we had finished a pilot screen to identify chemically induced mutations affecting X. tropicalis early development.  However, one of the most striking mutants appeared not in the ENU screen, but while feeding a tank of tadpoles from a transgenic line that we were breeding to homozygosity for an unrelated project. As the tadpoles began metamorphosing into froglets about 25% of them developed without arms (see attached picture, courtesy T. Geach), and it became increasingly likely that we were looking at the result of a serendipitous transgene insertion disrupting a locus that was necessary for forelimb development.  The effect of the insertional mutation appeared to be very specific, no other developmental defects were evident.

I was able to fairly rapidly clone the site of insertion in the nephronectin (npnt) gene, which had been recently identified in Louis Reichardt’s lab as a ligand for a8b1 integrin, necessary for metanephros development in mouse.  Thus the xenopus de milo (xdm) mutant suggests a novel role for integrin signalling in limb development.  We are lucky to work in a large, interdisciplinary institute, in the same division as Malcolm Logan, an expert on limb type specification and patterning, and a collaboration was quickly set up with Satoko Nashimoto, a postdoc in the Logan lab.  Working together we were able to show that xmd homozygotes did not express tbx5, the earliest marker of the emerging forelimb, in the forelimb field, never developed forelimb buds, and that all elements of the forelimb skeleton were absent in xdm froglets, consistent with a role for npnt in forelimb initiation.

Amphibian limb regeneration has been extensively studied but little is known about limb initiation in Xenopus.  Frogs develop arms after several weeks as free-swimming tadpoles, as opposed to other vertebrates in which limbs form in parallel with other organ systems relatively early in embryogenesis.  There is some evidence that the specific lineages and signalling pathways necessary for limb initiation in Xenopus are different from those in chick or mouse.  Significantly the mouse npnt knockout does not have a forelimb phenotype.  It remains to be determined whether integrin signalling is necessary for forelimbs uniquely in frogs, or whether it plays a more general role, but is mediated through other ligands in mammals.  Understanding how the metamorphosing tadpole initiates limbs that are highly homologous to those of other vertebrates will help us understand processes critical for vertebrate limb formation and regeneration.

(15 votes)

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It’s been a busy time for me at ASCB, held this year in Philadelphia. As a long standing member of the Women in Cell Biology (WICB) committee, I have been part of a community of men and women interested in issues of career development for junior scientists in the life sciences. On Saturday, we held a workshop called “Leveraging your PhD in the real world” and yesterday we held our annual career discussion and mentoring roundtables. As usual, I hovered over the “scientific editing” and “scientific writing” roundtables, and am grateful to all the editors and writers who gave of their time to mentor young scientists. Then last night – sponsored by the Public Information Committee, there was a fascinating discussion moderated by Rex Chisholm and including Nobel laureates Eric Wieschaus and Marty Chalfie and future ASCB president Sandy Schmid about secrets to their success – discoveries they were most proud of, the importance of going to seminars and talking about “half-baked” (to quote Eric Wieschaus) ideas, and knowing when to stop. Tonight are the WICB junior and senior awards and the mentoring theater. Congratulations to junior awardee Magdalena Bezanilla and senior awardee (DMM editorial board member) Zena Werb!

ASCB is very welcoming of developmental biologists and there have been many prominent ones in attendance. It’s an international and inclusive organization, and I encourage you to consider joining the society and adding the ASCB meeting to your busy calendars.

(2 votes)

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We can all articulate the importance of using model organisms to understand biology, but many of us fall short in our understanding of some of the more uncommon model organisms.  Thankfully, there are amazing biologists out there that save the day!  These researchers use some of the more atypical model organisms to understand how different organisms develop and how developmental processes have evolved.  Today’s image features the crustacean Parhyale hawaiensis.

The establishment of the dorsoventral (DV) axis in many organisms is fundamental to the proper organization of organs and tissues.  In arthropods, the organization of tissues around the DV axis is well conserved, yet how the axis is established is not.  For example, ventral midline cells play a restricted role in DV patterning in Drosophila, yet they play a prominent role in establishing the DV axis in the crustacean Parhyale hawaiensis, according to a recent paper by Vargas-Vila and colleagues published earlier this year in Development.  In addition, the Parhyale ortholog of the transcription factor gene single-minded (Ph-sim) is expressed in midline cells and is required for differentiation of midline cells.  These results suggest the importance of ventral midline cells in DV patterning in the last ancestor common to both crustaceans and insects.

Images above show Parhyale embryos at different stages, with nuclei in blue.  Throughout early development, Ph-sim is expressed in ventral midline cells, as seen in the false-color overlay (red) of expression patterns (A-C).  In Ph-sim (RNAi) embryos, midline staining of the midline marker Ph-otd-1 is absent (compare D and E), and the nice clear line of ventral midline cells (red arrows in F) is no longer visible (compare F and G).

Vargas-Vila, M., Hannibal, R., Parchem, R., Liu, P., & Patel, N. (2010). A prominent requirement for single-minded and the ventral midline in patterning the dorsoventral axis of the crustacean Parhyale hawaiensis Development, 137 (20), 3469-3476 DOI: 10.1242/dev.055160

(8 votes)

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Scientists don’t spend free time to think about the changes that made possible the birth of a new way to make research. For example, how we moved from a world driven by religious and philosophical beliefs to a world demanding explanations and mechanisms? Ernst Haeckel was one of the scientists who made that change possible and, more important to us, he accomplished it from the field of developmental biology. Although this post is not intended to be a review of Haeckel’s work (that would be a daunting and overwhelming task), it is worth to mention some interesting things about this story. As many scientists at their times, Haeckel’s work was criticized and Haeckel was, indeed, heavily attacked not only by religious and conservative people, but also by other fellow scientists. We can summarize some aspects of Heckel’s view as follows: Haeckel was impressed and inspired by the work of previous scientists (being J.F. Meckel and K.E. von Baer the most important people), showing the resemblance between embryos from different animal species at early stages of development. Although Haeckel was not the first scientist to propose a resemblance of vertebrate embryos at early stages of development, Haeckel made use of this fact, often depicting embryos in his works with some degree of abnormalities (which were used by his critics to accuse him of adulterating embryos).

Ernst Haeckel formulated the known Fundamental Biogenetic Law, in which he describes the parallelism between embryonic development and the phylogenetic history, claiming that embryonic development is a rapid recapitulation of the evolution, or “ontogeny recapitulates phylogeny”. Most conservative people viewed Haeckel’s propositions as a challenge to the more religious views about the origin of man. Haeckel made comparisons between early embryos from different species; his famous drawings, that appeared in his works, especially in Natürliche Schöpfungsgeschichte, were famous at the time, and they were criticized by other scientists. Some people at the time claimed that the only evidences for this proposition were the drawings made by Haeckel, but we have to consider that experimental biology was at a sort of “very early stage of development”. Hence, Haeckel’s work was abandoned from the main stream of science, especially between the World Wars, when chemistry and physics gained much more attention. However, Haeckel’s work likely inspired many future scientists, including his students. One of them was Hans Spemann, who later made one of the most important experiments in biology. Even, when the findings of Spemann and Mangold can be considered as opposed to Haeckel’s biogenetic law (because now the embryological development is driven by hidden forces with molecular nature, and since all organisms are different, these forces should differ in nature), the work of Spemann led in time to the discovery and (partial) understanding of the Wnt pathway, which is maybe one of the most conserved signaling pathways in nature and one of the most important driving forces in embryological development, validating Haeckel’s work: indeed, embryological development involves the expression and function of conserved genes through evolution. This realization brought Haeckel’s work one more time into the public attention, and once again, critics to his work appeared, with high press coverage at the time.

Image attributed to Ernst Haeckel, published in his work  Natürliche Schöpfungsgeschichte, and illustrates the similarities between embryos of different species (man, dog and turtle). His rivals argued that embryos compared in Haeckel’s drawings usually had abnormalities and that they corresponded to different developmental stages. Image source: Wikipedia Commons.  

Today, two papers published in Nature (vol. 468, Number 7325) “recapitulate” this classic debate: Domazet-Lošo and Tautz show in Zebrafish that the transcriptome expressed during the phylotypic stage (the stage in which species from a phylum resemble each other) is older compared with the transcriptome expressed in adult stages. They conclude that “our study provides strong molecular support for a correlate between phylogeny and ontogeny”, which agrees with the propositions of Haeckel and previous researchers (like K. von Baer). In the same issue, Kalinka and co-workers took a similar approach with six Drosophila species, observing also maximal conservation of gene expression at the phylotypic stage. Haeckel was discredited by many scientists, even in these days. He has been accused to be convinced to fraud, showing that embryos in drawings are stylized, altering embryos, and heterochrony is not considered in the drawings itself. With the available tools nowadays, we know that embryological development is variable between species. I believe that this is not the point. Haeckel’s work helped to popularize an important idea in biology, and we can discuss at which extent conserved genes and signaling pathways are integrated in the early (or late) development, validating the general concept about the relationship between evolution and development.

It should be an outstanding improvement in scientific journals, to include historical profiles and short reviews (no more than one page) about these relevant figures in biology (and science in general).

(5 votes)

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