This cartoon was first published in the Journal of Cell Science. Read other articles and cartoons of Mole & Friends here.

Part I- ‘The imposter’

Part II- ‘The teaching monster’

Part III- ‘The Pact’

Part IV- ‘The fit’

Part V- ‘The plan’

Part VI- ‘FCTWAWKI’

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The laboratory of Sarah Goetz at Duke University School of Medicine is inviting applications for a postdoctoral researcher.

Our group studies the biology of the primary cilium. We are working to identify the signaling pathways that control the assembly of this essential organelle, as well as to define the roles of primary cilia in embryonic development and in human diseases. We use cell culture as well as mouse models to explore these questions.

Ideal candidates will be accomplished, highly motivated, and creative scientists with a recent Ph.D. in the life sciences (less than 2 years of postdoctoral experience is strongly preferred), or who anticipate completion of their degree prior to starting the position. Previous experience in cell biology, neurobiology and/or mouse genetics is desirable. Applicants should email a brief cover letter describing research accomplishments and future research goals, current CV with list of publications, and contact information for 3 professional references to:

sarah.c.goetz@duke.edu

For more information, see www.goetzlabduke.com

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Tunicates are the invertebrates most closely related to us, forming a monophyletic clade with the vertebrates, known as Olfactores. Tunicates, often erroneously referred to as “urochordates” (a junior synonym and thus a taxonomically invalid term), have revealed many insights into the development and evolution of chordate- or vertebrate-specific tissues and organs, such as the notochord, thyroid, liver, craniofacial muscles, heart, spinal cord, and more. Of course, one of the most important vertebrate features is a population of cells called the Neural Crest Cells (NCCs). In all chordates, the central nervous system derives from a dorsal neural plate, which rolls into a dorsal hollow neural tube. In vertebrates, NCCs are specified all along the lateral borders of the neural plate, which meet at the dorsal midline upon neural tube closure. There, NCCs undergo an epithelial-to-mesenchymal transition to form what’s been sometimes called the “Fourth Germ Layer”¹. This is because NCCs are a population of stem cell-like progenitors that delaminate and migrate to give rise to a dizzying array of cell types all throughout our bodies and most of the skull: pigment cells, sensory neurons, glia, cartilage, bone, connective tissue, smooth muscle, and chromaffin cells of the adrenal medulla.

Because NCCs are so special to us, there has been much interest in understanding its evolutionary origins. There have been many theories and many disagreements, but a clear picture is starting to emerge. One of the biggest clues pointing to the origin of NCCs is one derivative cell type in particular: pigment cells containing melanin, or melanocytes. In cephalochordates, the other major chordate subphylum, cells along the lateral borders of the neural plate give rise to melanocytes associated with a series of light-sensing organs in the neural tube, known as Dorsal Ocelli². These are not to be confused with the melanocytes of the frontal eye, which appear to be homologous to the pigment cells of the vertebrate retina instead³. In tunicates, similar dorsal melanocytes arise from the lateral borders of the neural plate and become associated with a light-sensing ocellus and a gravity-sensing otolith⁴. Recently, the gene regulatory networks specifying and differentiating these cells have been shown to be shared with the NCC-derived melanocytes of vertebrates, strengthening the case for homology⁵. Therefore, the latest models of NCC evolution propose that the neural plate borders of the pre-vertebrate ancestor already gave rise to one NCC derivative: melanocytes. Presumably, the ability to delaminate, migrate, and differentiate into several different cell types would have been added on to these ancestral melanocyte progenitors^6,7.

The major questions stemming from this model are: how did the vaunted multipotency of the NCCs evolve? How did they acquire their amazing migratory capabilities? Were these traits coupled to a singular evolutionary co-option event, or have they been added on, piecemeal, over the course of chordate evolution? To begin answering these questions, one must look carefully at all those other derivatives of vertebrate NCCs Some, like cartilage and bone, are clearly a co-option of an ancestral mesoderm derivative⁸. Other cell types are likely vertebrate-specific, like chromaffin cells. However, the picture is less clear when looking at NCC-derived neurons, since clear homologs of these have not been found in tunicates or cephalochordates. Although the peripheral nervous systems of tunicate larvae have several sensory neuron subtypes⁹, none of them have been decisively linked to NCCs, either because they do not arise from the neural plate borders or because they more closely resemble non-NCC-derived sensory cells in vertebrates.

Here I describe and comment on our recent results¹⁰, which have further refined our models of NCC evolution. We were intrigued by a particular neuronal subtype that had been recently discovered and described in the tunicate Ciona intestinalis, named the Bipolar Tail Neuron (BTN)⁹. So called due to their bipolar, or more accurately, pseudo-unipolar morphology, bearing a single axon divided into two long neurites, or processes: an anterior, proximal process extending towards the head of the tadpole, and a posterior, distal process extending to the tip of the tail. We showed that the BTNs receive synaptic inputs from sensory cells embedded in the epidermis of the tail¹¹. Furthermore, we also found that the BTNs synapse onto neurons of the Motor Ganglion, the spinal cord homolog of tunicates. Thus, the BTNs form a relay, part of a neural network that transmits sensory inputs from the periphery to the central nervous system.

This configuration is reminiscent of certain sensory pathways in vertebrates, in which NCC-derived Dorsal Root Ganglia (DRG) neurons relay peripheral sensory inputs from epidermal neurons known as Merkel Cells¹², to neurons of the spinal cord and beyond. The shared molecular features are also intriguing, as both DRG neurons and BTNs express the transcription factor genes Neurogenin (Neurog) and Islet ^13-15, and mechanosensation-modulating Acid-Sensing Ion Channels (ASICs)¹⁶. These parallels prompted us to wonder whether the BTNs, like the melanocytes of Ciona, could represent an evolutionary forebear of the NCCs. We reasoned that further support for this possibility would come from identifying the embryological origins of the BTNs (whether they originate at the borders of the neural plate), and characterizing their development (whether they engage in delamination and migration).

Since we knew that the BTNs express Neurog, we looked at the expression of this gene by mRNA in situ hybridization, and found that it is activated in two lateral rows of cells at the very posterior end of the embryo at the early neurula stage (Figure 1). We showed that these cells are at the boundary between the epidermis and neural tissue, and express Msx and Pax3/7, conserved markers of the neural plate borders in all chordates. In vertebrates, NCCs arise from Msx⁺/Pax3/7⁺ neural plate border cells, and in fact these genes encode upstream regulators of many NCC specification genes¹⁷. Using a Neurog reporter gene to follow these cells throughout development, we found that BTN precursors delaminate shortly after neural tube closure, and migrate as a simple chain of two cells on either side of the neural tube (Figure 2). We found that they migrate outside the neural tube, along paraxial mesoderm, evocative of the migration of NCC-derived DRG neuroblasts along paraxial mesoderm on either side of the vertebrate neural tube. As the BTNs migrate anteriorly to the middle of the tail, their intrinsic polarity is reversed and begin to extend their posterior processes, suggesting a precisely timed re-orientation of cell polarity underlies their distinctive bipolar morphology.

We also investigated the molecular control of BTN specification and differentiation, and found that this is under the control of Neurog itself, which in turn is tightly regulated by FGF/ERK signaling. While we are still working to sort out these steps in detail, our current findings as they stand have some interesting implications for models of NCC evolution. Mainly, we present evidence that the BTNs and the DRG neurons might be homologous, which would imply that last common ancestor of vertebrates and tunicates might have had neural plate borders capable of giving rise to more than one NCC derivative cell type, in this case, pigment cells and sensory relay neurons. Furthermore, our observation of the delamination and migration of the BTNs along the paraxial mesoderm suggests that some aspects of NCC migratory behavior may also pre-date the emergence of vertebrates and bona fide NCCs.

Our new model, building on previous models, posits that the olfactorean ancestor already had neural plate borders capable of generating both pigment cells and sensory relay neurons, which in vertebrates are two derivatives of NCCs. In the highly reduced tunicate embryo, pigment cells and sensory neurons arise from opposite ends of the neural plate borders. However, recall that cephalochordates have serial pigmented dorsal ocelli, so perhaps the original chordate ancestor generated multiple pigment cell and sensory neurons all along the neural plate borders. In this model the real novelty of vertebrate NCCs is not the ability to give rise to multiple cell types, which in our model is an ancestral character. Instead, the key difference would have been the intercalation of a multipotent stem cell-like regulatory program within the ancestral network, downstream of neural plate border specification and upstream of multiple cell type differentiation programs¹⁸. This multipotent progenitor state, a hallmark of NCCs, allowed for these cells to undergo a prolonged period of delamination, migration, proliferation, and more dynamic modes of downstream cell fate specification and differentiation. In sum, our findings lend further support to the longstanding idea that vertebrate NCCs were not an abrupt vertebrate innovation, but rather derived from a set of ancestral features, elaborated over the course of chordate evolution¹⁹.

1              Hall, B. K. The neural crest as a fourth germ layer and vertebrates as quadroblastic not triploblastic. Evolution & development, 3-5 (2000).

2              Yu, J.-K., Meulemans, D., McKeown, S. J. & Bronner-Fraser, M. Insights from the amphioxus genome on the origin of vertebrate neural crest. Genome research 18, 1127-1132 (2008).

3              Vopalensky, P. et al. Molecular analysis of the amphioxus frontal eye unravels the evolutionary origin of the retina and pigment cells of the vertebrate eye. Proceedings of the National Academy of Sciences 109, 15383-15388 (2012).

4              Nishida, H. & Satoh, N. Determination and regulation in the pigment cell lineage of the ascidian embryo. Developmental Biology 132, 355-367 (1989).

5              Abitua, P. B., Wagner, E., Navarrete, I. A. & Levine, M. Identification of a rudimentary neural crest in a non-vertebrate chordate. Nature 492, 104-107 (2012).

6              Wada, H. Origin and evolution of the neural crest: a hypothetical reconstruction of its evolutionary history. Development, growth & differentiation 43, 509-520 (2001).

7              Ivashkin, E. & Adameyko, I. Progenitors of the protochordate ocellus as an evolutionary origin of the neural crest. EvoDevo 4, 12 (2013).

8              Jandzik, D. et al. Evolution of the new vertebrate head by co-option of an ancient chordate skeletal tissue. Nature 518, 534-537 (2015).

9              Imai, J. H. & Meinertzhagen, I. A. Neurons of the ascidian larval nervous system in Ciona intestinalis: II. Peripheral nervous system. The Journal of comparative neurology 501, 335-352 (2007).

10           Stolfi, A., Ryan, K., Meinertzhagen, I. A. & Christiaen, L. Migratory neuronal progenitors arise from the neural plate borders in tunicates. Nature 527, 371-374 (2015).

11           Torrence, S. A. & Cloney, R. A. Nervous system of ascidian larvae: caudal primary sensory neurons. Zoomorphology 99, 103-115 (1982).

12           Maksimovic, S. et al. Epidermal Merkel cells are mechanosensory cells that tune mammalian touch receptors. Nature 509, 617-621 (2014).

13           Ma, Q., Fode, C., Guillemot, F. & Anderson, D. J. Neurogenin1 and neurogenin2 control two distinct waves of neurogenesis in developing dorsal root ganglia. Genes & development 13, 1717-1728 (1999).

14           Sun, Y. et al. A central role for Islet1 in sensory neuron development linking sensory and spinal gene regulatory programs. Nature neuroscience 11, 1283-1293 (2008).

15           Stolfi, A. & Christiaen, L. Genetic and genomic toolbox of the chordate Ciona intestinalis. Genetics 192, 55-66 (2012).

16           Coric, T., Passamaneck, Y. J., Zhang, P., Di Gregorio, A. & Canessa, C. M. Simple chordates exhibit a proton-independent function of acid-sensing ion channels. The FASEB Journal 22, 1914-1923 (2008).

17           Simões-Costa, M. & Bronner, M. E. Establishing neural crest identity: a gene regulatory recipe. Development 142, 242-257 (2015).

18           Bronner, M. E. & LeDouarin, N. M. Development and evolution of the neural crest: an overview. Developmental biology 366, 2-9 (2012).

19           Baker, C. V. & Bronner-Fraser, M. The origins of the neural crest. Part II: an evolutionary perspective. Mechanisms of development 69, 13-29 (1997).

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The Alberto Monroy Fellowship is awarded to an Italian citizen to support their attendance in the Embryology Course next summer at the Marine Biological Laboratory in Woods Hole, Massachusetts. The amount of the fellowship is 5,500.00 euros (about $6,000.00). The full text of the announcement of the award competition for 2016 can be found at http://www.giomolab.it/AAM/bando%20Monroy%202016.pdf. The deadline for applications is February 1, 2016.

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Applications are invited for a postdoctoral position at the University of California San Francisco, Broad Center of Regeneration Medicine and Stem Cell Research, in the laboratory of Dr. Katja Brückner.

The laboratory investigates molecular principles of how the peripheral nervous system and its inputs regulate stem cell niches and tissue microenvironments during animal development and homeostasis. We utilize established Drosophila melanogaster models that address the regulation of hematopoiesis by sensory neurons and extrinsic stimuli. The lab also aims to expand the research program into other organ systems in Drosophila, and complementary systems of murine stem cells and self-renewing tissue macrophages. Projects are federally funded by NIH R01 and other NIH and NSF grants.

The ideal candidate is a talented, determined and creative scientist with a solid background in developmental biology and genetics. Prior work with Drosophila genetics, invertebrate or vertebrate neurobiology or hematopoiesis/macrophage biology is preferred. General basic skills in molecular biology, histology and cell-based assays are expected. Candidates must hold a recent PhD, MD or MD/PhD degree (or anticipate such a degree prior to starting the postdoc), have prior publications and be motivated and hardworking.

UCSF and the Broad Center offer a vibrant scientific environment, competitive salary and benefits. Please send a CV, statement of research interests and names and contact information of three references to: katja.brueckner@ucsf.edu

http://bruecknerlab.ucsf.edu/

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The three-pound lump under our skulls that allows us to speak, run and function in our daily lives is a mass of dozens of types of minuscule cells joined in an intricate web of communication. We pop out of the womb with our brains ready for us to interact with the world, but throughout our lives, new cells are born and others die, connections form and wither. There is a group of cells that makes this possible. They are our brain’s store of stem cells, which can become all of the different types of cells of the brain, including neurons and glial cells.

The brain’s stash of stem cells is important for both maintenance and healing after injury. Some of them become neurons, others glial cells, and some remain as the trusty store of stem cells. When cells change from the stem cell version to a specific type of cell, this is called activation (in scientist lingo). Ana Martin-Villalba and her team of researchers at the German Cancer Research Center wanted to know what makes a stem cell change from the dormant state to this activated one.

They looked at the SVZ (subventricular zone), which is a part of the brain with a hub of stem cells. Using human cells, they looked at the genes expressed in individual cells. Let that one sink in. We can study the genetics of separate cells, and taken together look at the gene expression of whole groups of cells. Based on the gene expression, they distinguished two groups, quiescent and active. The quiescent cells were broken down further into dormant and primed cells, which are ready to become active. The researchers then compared the dormant and active cells, to see how they acted differently. They found that the dormant cells were churning out proteins, the molecules that cells use to function and communicate, whereas the active cells were busy specifying more into specific cell types (a process known as differentiation).

This month’s image looks like a mosaic with each tile representing a single gene in a single stem cell. The cells in this grid were undergoing a simulated stroke, receiving decreased blood flow and oxygen. Simulating this physiological perturbation and examining the response of the stem cells showed that just as there are subsets of cells under normal conditions, subsets of cells respond differently under injury. Not only that, the response showed activation. This study solves a piece of the puzzle of how the cells are being stimulated to become active. A molecule called interferon gamma helps orchestrate the transition.

Understanding the stem cells in our brain can have important implications for helping our brains heal after brain injury or in neurodegenerative disorders.

Related content

Stem cells and neurological disorders – topic page

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The use of green fluorescent protein (GFP) has revolutionised the study of dynamic cellular processes in cells, tissues, and whole organisms. Laboratories throughout the world have exploited the simplicity of GFP as an everyday tool to determine protein localisation in cell lines and whole organisms. Fluorescence microscopy now dominates the imaging field although the attainable resolution of these methods has always been a limiting factor. Alternative techniques such as electron microscopy offer higher resolution but have traditionally been viewed as slow and technically difficult.

An electron microscopic method that provides rapid, simple, high resolution, and quantitative detection of GFP-tagged proteins would represent a significant advance in the fields of cell and developmental biology. Ideally this method would be compatible with the new 3D electron microscopic techniques now available, including electron tomography and serial blockface scanning electron microscopy and not require new or highly specialised equipment. We have now developed a new technique that we believe fulfils all these requirements (Ariotti et al, 2015).

We have shown that we can target a recently developed modified peroxidase, called APEX (Martell et al, 2012), to any GFP-tagged protein of interest by co-expression with a GFP-binding peptide directly attached to the APEX-tag (Ariotti et al, 2015). The APEX peroxidase produces an electron dense reaction product at the site of the GFP-fusion protein that allows its visualisation in the electron microscope. With this method whole libraries of GFP-tagged proteins could be localised at electron microscopic resolution in a few days. Moreover, the method was shown to generate remarkably high-resolution localisation data. We can localise proteins to distinct subdomains of microdomains of subcellular organelles such as individual intraluminal vesicles within endosomes and specific protein subdomains of the neck region of caveolae. This method proves to be a very powerful and versatile technique for EM analysis. It is quantitative by linescan analysis, compatible with tomography and serial blockface electron microscopy for 3D analysis of whole cell protein distribution, and offers a simplistic alternative to involved correlative light and electron microscopy techniques that are currently available in the literature.

Perhaps of most interest to developmental biologists is the application of this technique to localisation of GFP-fusion proteins in whole organisms (Ariotti et al, 2015). The effort and cost required to generate entirely new and separate gene edited lines for fluorescence and electron microscopic analyses (by conventional APEX tagging methods) are prohibitive. We bypassed this bottleneck by generating new zebrafish lines that express APEX-GFP binding peptide in all tissues. We designed two different zebrafish lines under the control of two different promoters. The first line exploits a beta-actin2 promoter for ubiquitous and continual expression of GFP-binding peptide-APEX fusion in all cells. The second zebrafish line allows for temporal control of the expression of the EM-marker, as it was engineered under the control of the hsp70I promoter that, after a brief and mild heat shock, induces rapid expression and accumulation of the APEX-GFP binding peptide in the cytoplasm of all cells within in the animal. With a single cross between our gene edited lines and any zebrafish stably expressing a GFP-tagged protein we could show rapid electron microscopic localisation of proteins including those expressed at endogenous levels in vivo. By the exploitation of the hsp70I promoter it will be possible to track subcellular protein distribution changes during development to a resolution of approximately 10 nm rapidly and easily. To further simplify the system, both lines were engineered with a red lens to denote expression of GFP-binding peptide-APEX fusion protein for easy genotyping of progeny.

We envisage that this method will provide a rapid localisation strategy in cells and whole organisms. It does not require new or expensive EM equipment, but rather is adapted from an established protocol that has been standard in the electron microscopy field for decades. While we have only demonstrated the utility of the method in zebrafish embryos the technique will not be limited to this particular animal model and we predict will be of use in other species.

References

Ariotti N., Hall T.E., Rae J., Ferguson C., McMahon K.A., Martel N., Webb R.E., Webb R.I., Teasdale R.D., and Parton, R.G. (2015). Modular detection of GFP-labeled proteins for rapid screening by electron microscopy in cells and organisms. Dev. Cell. 35, 513-25.

http://www.ncbi.nlm.nih.gov/pubmed/26585296

Martell, J.D., Deerinck, T.J., Sancak, Y., Poulos, T.L., Mootha, V.K., Sosinsky, G.E., Ellisman, M.H., and Ting, A.Y. (2012). Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat. Biotechnol. 30, 1143-1148.

http://www.ncbi.nlm.nih.gov/pubmed/23086203

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Happy holidays everyone!

It’s almost the New Year! The Node will be a bit quieter than usual for the next few days and we might take longer than usual in replying to messages.

But not to worry! If you’re craving some developmental biology, you can always have a look at our latest research posts. And if you still need to find a few last minute presents, make sure to check Cat’s science gift ideas, and let us if you’ve found anything that you’d like to add!

The New Year will bring with it new research, so why not share your 2016 research wish list with us? And while you’re at it, have some fun (and find out how far in development you are) with the Node’s DevBio Quiz!

Happy holidays and a happy New Year from the Node!

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In October, I attended the latest Company of Biologists Workshop “Transgenerational Epigenetic Inheritance“. Marco Geigges, one of the attendees at the Workshop, has already posted his impressions of the event. While there, I had the opportunity to speak to the organisers and some of the speakers and early career scientists, and we recorded this short movie about the Workshop. We hope this gives you a flavour of the event, and we encourage you to keep an eye out for future Workshops that you might be interested in attending!

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A position (#121682) is available immediately for a Research Technician/Faculty Specialist to contribute to our studies in neural crest and placodes. The Technician will conduct research, assist in the training of students, and take part in the management of the laboratory of Dr. Lisa Taneyhill at the University of Maryland.

Laboratory skills should include the ability to perform various molecular biology and biochemical assays, such as recombinant DNA/cloning; immunoprecipitation and immunoblotting; and/or immunohistochemistry. Experience with microscopy, chick embryology, and tissue culture is desirable. For more information on the lab, please see http://www.ansc.umd.edu/people/lisa-taneyhill.

Qualifications: An Bachelor’s degree (B.A. or B.S.) in a related field and prior laboratory research experience is essential. Fluency in spoken and written English is required.

Compensation: Salaries are highly competitive, negotiable and commensurate with qualifications. Fringe benefits offered.

Applicants must apply through eTerp at https://ejobs.umd.edu. Applications will be accepted until a suitable candidate is identified.

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