Amelia Lane, David A. Parfitt, Conor M. Ramsden, Peter J. Coffey, Michael E. Cheetham

Parfitt et al. Cell Stem Cell 1–13 (2016)

Making eyes

Methods to differentiate human stem cells into retinal cell types have been under development for almost a decade. Stem cell derived retinal cells provide a rich resource to study unique and otherwise inaccessible cells. Reprogramming somatic cells into iPSC allows us to study retinal cells carrying pathogenic mutations that cause retinal degeneration. This enables the interrogation of disease mechanisms and allows us to screen mutation-specific therapies such as antisense oligonucleotides on patient cells. The substantial technical challenges of consistently and uniformly generating terminally differentiated photoreceptors are gradually being overcome allowing us to make use of this new and powerful experimental paradigm.

The retina is derived from the anterior neural plate. During vertebrate eye development a common pool of progenitor cells invaginate to form a double walled optic cup. The cup is patterned by interactions with the adjacent surface ectoderm to form neural retina (NR) and an overlying monolayer of pigmented cells known as retinal pigment epithelium (RPE) (Fuhrmann et al., 2000). Mature photoreceptors in the human retina have a unique structure suited to their purpose as sensors that transduce light into an electrochemical signal. Rod and cone cells have distinct compartments. Their cell bodies are arranged in an outer nuclear layer (ONL), consisting of tightly packed columns approximately 10 nuclei in thickness. At the apical edge they have tight junctions forming an outer limiting membrane (OLM). Above the OLM are inner-segments (IS), which house much of the ER and Golgi and are densely packed with mitochondria. Protruding from the IS of each photoreceptor cell is a specialised cilium, the outer segment (OS), a 25um stack of membranous discs packed with photosensitive opsin proteins and the phototransduction machinery. The OS is joined to the IS by a transition zone referred to as the connecting cilium (CC).

The first differentiated retinal cell type to be derived from human embryonic stem cells (hESC) were RPE, the cells which overlay the photoreceptors in the retina. Pigmented RPE were first detected following spontaneous differentiation of confluent hESC cells (Klimanskaya et al 2004). It has since been demonstrated by several labs around the world that induced pluripotent stem cells (iPSC) and hESC will spontaneously differentiate into RPE within a month. Making photoreceptors requires longer periods of time similar to the process in utero. In the first published attempts at photoreceptor differentiation, hESC cells were treated with Wnt and nodal inhibitors followed by long term treatment (more than 130 days) with retinoic acid and taurine (Osakada et al., 2008, Lamba et al., 2006). These protocols generated immature photoreceptor progenitor cells and/or opsin-expressing cells with no specific morphological resemblance to photoreceptors in a two-dimensional format.

Pioneering work by the Sasai lab at the RIKEN Centre for Developmental Biology in Japan, using a three dimensional ‘organoid’ approach for cell differentiation, greatly improved the quality of the photoreceptor cells derived. First with mouse, then with hESCs, Sasai’s lab was able to create photoreceptors from embryoid bodies (EBs) that formed ­­­three-dimensional (3D) ‘optic cups’ with RPE and several neural retina layers (Eiraku et al., 2011, Nakano et al., 2012). This was rapidly followed by similar demonstrations in human iPSC (Zhong et al., 2014, Reichman et al., 2014). These opsin-expressing photoreceptor cells developed mitochondria-rich inner segments with cilia easily observed, raising the tantalising possibility that they may be capable of generating OS in vitro.

Disease modelling

Our lab’s research interest in inherited retinal dystrophy disease mechanisms prompted us to attempt to make 3D retinas from cells donated by a patient with a type of retinal degeneration known as Leber congenital amaurosis (LCA). LCA is a recessively inherited retinal dystrophy resulting in severe visual loss in early childhood. This patient is homozygous for a deep intronic mutation in the gene encoding CEP290 (c.2991+1665A>G), which is the most common LCA associated allele. CEP290 is found at the transition zone of all ciliated cells and in the CC of photoreceptors. Its position indicates a role in formation of the Y-shaped linker structures that anchor the ciliary axoneme to the plasma membrane. Mutations in CEP290 can cause syndromic ciliopathy involving several organs or LCA alone. The common c.2991+1665A>G mutation is only associated with LCA and leads to mis-splicing of the CEP290 transcript and inclusion of the stop-codon containing cryptic exon. Some correctly spliced transcript remains and it is thought that the residual protein level is sufficient to prevent a syndromic ciliopathy in affected individuals. Fibroblasts and lymphoblast cultures derived from these patients display a cilia defect; with reduced numbers of shortened cilia (Collin et al., 2012, Gerrard et al., 2012, Garanto et al., 2016). In patients however, it is not clear why retinal cells appear to be affected more than other tissues.

Our first attempts at the 3D organoid method used embryonic stem cells (ESC) and control iPSC derived from neonatal foreskin fibroblasts. The ESC EBs generated very promising bi-laminated optic cups with retinal ganglion and neuroblastic cell layers as well as RPE. The neuroblastic layer contained photoreceptor progenitors, but we did not continue the differentiation for long enough to generate mature photoreceptors with IS.

We then began large scale and long-term differentiations using both control and CEP290 patient derived iPSC lines. We tried both Zhong and Nakano protocols from several patient and control lines and carried out immunocytochemistry (ICC) and gene expression analysis to follow the time course of photoreceptor differentiation.

CEP290 patient fibroblasts reprogrammed efficiently into iPSC. Similar to the fibroblast cells, there was a significant reduction in ciliation in iPSC; however, this did not appear to affect their pluripotency. CEP290 iPSC efficiently differentiated into early derivatives of all three germ layers. We found that different clonal lines from both control and patient iPSC formed EBs with varying efficiency (50-100%). Of these, a proportion could be seen to generate transparent, radially aligned neuroepithelium (NE) – which again varied between lines (5-30%). We were able to distinguish these NE-producing EBs morphologically with a light microscope and dissect out the neuroepithelial buds (Figure 1). Unfortunately, a proportion of these did not survive the dissection procedure or spontaneously collapsed and became necrotic. However, the precious surviving organoids were cultured for up to 21 weeks.

We processed EBs early on in the differentiation process and saw that the basal bodies and associated cilia had aligned at the apical surface of the EBs with neuroepithelial domains, thereby fulfilling their roles as sensors that detect and relay signals from the outside environment, and facilitating polarised cell division. Excitingly we detected a phenotype in our CEP290 EBs – significantly fewer of the pericentrin positive basal bodies at the apical surface possessed ARL13B positive cilia. This did not appear to reduce the efficiency with which CEP290 EBs generated organised neuroepithelial domains; an interesting observation given the role of primary cilia in embryonic forebrain development (Willaredt et al., 2013).

We extracted RNA from individual EBs and analysed gene expression as well as cryosectioning them for ICC analysis. As the EBs developed it became clear that the differentiating cells were following the highly conserved sequence of eye development; similar to what has been observed in other studies of iPSC-retina differentiation (Meyer et al., 2009). Various photoreceptor cell markers such as recoverin and arrestin were being switched on over time (Figure 1). Comparing the time course over which markers were expressed it was clear that the patient cells were behaving similarly to controls and our model did not display any ‘developmental’ defects in vitro.

We used electron microscopy to analyse the ultrastructure of these developing photoreceptors; at week 13 tight junctions were visible all across the apical layer forming an OLM and above this mitochondria rich buds had appeared, with the morphology of inner segments.

At the latest time point (21 weeks), ICC revealed the presence of rhodopsin positive rods (Figure 2), as well as red/green and blue cone cells . These were arranged within a compacted apical layer, reminiscent of the ONL and approximately 5 nuclei thick. By this time, the interior of the cups was largely empty or necrotic. RPE cells could also be seen in several of the cups but it was generally adjacent to the photoreceptors cells and very rarely overlaying them as in the 2 walled optic cup seen in human development and reported by Sasai’s lab (Nakano et al., 2012).

Looking at this latest time point by electron microscopy we were astonished to see the presence of connecting cilia that were elaborating into partially stacked discs similar to rudimentary OS. The discs were aligned at the base but disorganised and broken at the tip. In vivo the outer segments are enveloped by the microvilli of the RPE and encased in an extracellular matrix with a unique composition known at the interphotoreceptor matrix (IPM) (Ishikawa et al., 2015). This proteoglycan-rich substance is secreted by both the RPE and the photoreceptors in vivo and, in contrast to the majority of ECM in other tissues, is lacking in a collagenous meshwork with neither laminin nor fibronectin as major components. We observed a lot of broken cilia and opsin positive debris at the apical edge and hypothesise that if we could place the developing photoreceptors in an environment that would stabilise the nascent OS, akin to the IPM, we might observe more organised and abundant OS structures. This could be a powerful tool for studying the numerous mutations in retinal dystrophy genes that lead to OS trafficking disorders and retinal degeneration.

There was a reduction in cilia number in CEP290 patient optic cups using ARL13B to mark the connecting cilia of opsin positive photoreceptors. In addition the connecting cilia were now significantly shorter than those in controls. However the most interesting insight came from looking at the relative levels of mis-splicing in the various cell types derived. While the ratio of correctly spliced: mis-spliced transcript in fibroblast, iPSC and iPSC-RPE was approximately 50:50, in photoreceptors the quantity of correctly spliced transcript was severely reduced down to only 10-20%. Cell type specific levels of mis-splicing could explain the specificity of retinal involvement characteristic of this particular mutation, with less full-length CEP290 protein in the retina than in other tissues. The reasons for this different mRNA processing are currently unclear, but it was recently shown that the human retina has unexpectedly high levels of splicing diversity (Farkas et al., 2013) and mutations in splicing factors can cause dominant retinal dystrophy. This finding raises the possibility that the retina could be especially sensitive to intronic variants that affect splicing.

Drug testing

The ability to make human retinal cells in vitro opens up lots of possibilities to study human development, disease mechanisms and test novel therapies. Antisense oligonucleotides (AON) that bind to and sterically block the aberrant splice donor site in the mutated CEP290 pre-mRNA have been developed and tested in patient fibroblasts (Collin et al., 2012 Gerrard et al., 2012). We were able to show that 10mm of an antisense morpholino (MO) specific to the LCA CEP290 mutation (CEP290-MO) transfected in EndoPorter solution (from Genetools) reduced the amount of cryptic exon containing-mRNA produced and increased ciliation in patient fibroblasts. We also showed that the treatment lasts up to 6 days.

Testing this potent MO on the CEP290 optic cups was the next logical step. In order to assess the capacity of the MO to enter the optic cup we used a fluorescein-tagged MO and imaged all layers of the cup 48 hours later using live confocal microscopy. The tagged MO was able to penetrate all layers of the optic cup and access the nuclei. Therefore, we treated 90-day-old patient optic cups with CEP290-MO every 3-4 days for 4 weeks and then assessed cilia length, cryptic exon expression, CEP290 levels and the amount of various CEP290 interaction partners at the basal body. CEP290-MO treatment significantly increased CEP290 protein levels, reduced cryptic exon expression and all but restored cilia numbers and length to wild type levels. Critically it also restored the localisation of the important CEP290 interacting partner, RPGR, to the connecting cilia. RPGR is located in the same ciliary compartment as CEP290 and mutations in RPGR are a major cause of retinitis pigmentosa (Breuer et al., 2002). We used high magnification confocal stacks to compare RPGR and CEP290 fluorescence at the ciliary base. We observed a significant depletion of RPGR and CEP290 in patient optic cups and a significant restoration following CEP290-MO treatment, an important functional read-out for the treatment efficacy.

In the absence of an animal model that accurately recapitulates the mis-splicing mutation, the patient iPSC-photoreceptor technique has yielded unique and important mechanistic insights and provided an explanation as to why this most common CEP290 mutation leads to a retina-only phenotype in human cells. In addition we have demonstrated the efficacy of AONs in rescuing correctly spliced CEP290 levels. Hopefully these studies will encourage the use of iPSC-derived organoids to study disease mechanisms and facilitate the development and clinical application of new therapies.

Breuer DK, Yashar BM, Filippova E, et al. A Comprehensive Mutation Analysis of RP2 and RPGR in a North American Cohort of Families with X-Linked Retinitis Pigmentosa. American Journal of Human Genetics. 70(6),1545-1554 (2002),

Collin, R. W. et al. Antisense Oligonucleotide (AON)-based Therapy for Leber Congenital Amaurosis Caused by a Frequent Mutation in CEP290. Mol. Ther. Nucleic Acids 1, e14 (2012).

Eiraku, M. et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472, 51–56 (2011).

Farkas, M. H. et al. Transcriptome analyses of the human retina identify unprecedented transcript diversity and 3.5 Mb of novel transcribed sequence via significant alternative splicing and novel genes. BMC Genomics 14, 486 (2013).

Fuhrmann, S., Levine, E. M. & Reh, T. a. Extraocular mesenchyme patterns the optic vesicle during early eye development in the embryonic chick. Development 127, 4599–609 (2000).

Garanto, A. et al. In vitro and in vivo rescue of aberrant splicing in CEP290 -associated LCA by antisense oligonucleotide delivery. Hum. Mol. Genet. (2016).

Gerard, X. et al. AON-mediated Exon Skipping Restores Ciliation in Fibroblasts Harboring the Common Leber Congenital Amaurosis CEP290 Mutation. Mol. Ther. Nucleic Acids 1, e29 (2012).

Ishikawa, M., Sawada, Y. & Yoshitomi, T. Structure and function of the interphotoreceptor matrix surrounding retinal photoreceptor cells. Exp. Eye Res. 133, 3–18 (2015).

Klimanskaya, I. et al. Derivation and Comparative Assessment of Retinal Stem Cells Using Transcriptomics. Cloning Stem Cells 6, 217-245 (2004).

Lamba, D. a, Karl, M. O., Ware, C. B. & Reh, T. a. Efficient generation of retinal progenitor cells from human embryonic stem cells. Proc. Natl. Acad. Sci. U. S. A. 103, 12769–74 (2006).

Meyer, J. S. et al. Modeling early retinal development with human embryonic and induced pluripotent stem cells. Proc. Natl. Acad. Sci. U. S. A. 106, 16698–703 (2009).

Nakano, T. et al. Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell 10, 771–85 (2012).

Osakada, F. et al. Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells. Nat. Biotechnol. 26, 215–24 (2008).

Willaredt, M. A., Tasouri, E. & Tucker, K. L. Primary Cilia and Brain Development. Mech. Dev. 130, 373–380 (2013).

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I am Yoshimasa Hamada, a Research Fellow in Okayama University Graduate School in Japan, working with Prof. Kenji Tomioka, Prof. Hideyo Ohuchi, Prof. Sumihare Noji and Dr. Tetsuya Bando. Our research focuses on the molecular mechanisms underlying leg regeneration, embryonic development, and circadian rhythm using the two-spotted cricket, Gryllus bimaculatus (Figure 1).

The two-spotted cricket, Gryllus bimaculatus

The two-spotted cricket Gryllus bimaculatus (De Geer) was newly described by Baron Charles de Geer in 1773. The latin name Gryllus means cricket, and bi-maculatus means two-spots, because Gryllus bimaculatus has a white spot in each forewing. The cricket has a global distribution, being found in European, African and Asian countries.  Gryllus bimaculatus (and the house cricket Acheta domesticus) is a popular food source of meat-eating arthropods, amphibians, reptiles and other pet animals. Hence, we easily buy the crickets from a pet shop at a low price (we can buy 5~10 crickets for £1). Interestingly, the cricket will be a diet even for us as well as for pets:  the Food and Agriculture Organization of the United Nations has proposed that insects would be a good food source for human. In fact, BBQ-flavored chips made from cricket flour are delicious for me!

The cricket is a hemimetabolous insect, with adults sized ~3cm. We easily feed them with goldfish chow and collect eggs using wet towel paper, and they hatch approximately two weeks after egg laying. Nymphs and adults are kept in the insect cages with folded paper as shelters (Figure 2). The growth rate depends on temperature: in standard breeding conditions (28 degrees Celsius), first instar nymphs hatch within two weeks, and nymphs grow up to adult by one month. All developmental stages are suitable for observation of detailed morphology, behaviors and gene expression during development.

Since crickets are relatively big and in comparison with other insects have strong vital force (which is suitable for dissection to detect action potential from targeted neurons), they have been used as a model insect for physiological research. Neuronal activities are measured by electrophysiological methods combined with pharmacological approaches.

In 1998, double-stranded RNA (dsRNA)-mediated interference (RNAi) was reported as a useful method for gene silencing by C. elegans researchers. RNAi is widely applicable for other organisms, including insects. Professor Noji’s group in Japan reported that RNAi is a powerful tool to analyze gene function during cricket embryonic development. His group also established transposon-based transgenics and genome editing techniques using zinc finger nucleases, TALE nucleases (TALEN) and CRISPR/Cas system (Nakamura et al., 2010; Watanabe et al., 2012), and analyzed whole genome sequence using next generation sequencing in Gryllus. These new methods established for the cricket open new avenues to understanding genetic basis of embryogenesis, regeneration, circadian rhythm, neurobiology, oogenesis, and so on.

Recently, we reported that epigenetic modifiers regulate leg regeneration (Development), embryonic development (Biology Open) and circadian clock (Zoological letters) by modulating specific gene expression via methylation on 27th lysine residue of histone H3. In this post, I introduce the biological features and benefits of the cricket, especially its use as a model animal in several scientific fields, e.g., regenerative and developmental biology, and chronobiology.

The cricket is a model insect of regeneration biology

Arthropods have a remarkable regenerative ability. The first scientific paper describing tissue regeneration was on the limb regeneration process of crayfish, written by René Réaumur in 1712. Hemimetabolous insects such as dragonflies, stinkbugs, grasshoppers and cockroaches were used for regeneration studies in 19^th and 20^th centuries. During the 1970s, the group of Prof. Vernon French documented limb regeneration and intercalary regeneration of the cockroach leg, and proposed a theory about positional values of organs; however, no molecular analyses were carried out on the cockroach. Among insect species, methods to analyze the molecular basis of organogenesis were only established in Drosophila melanogaster until the 1990s. However, the regenerative ability of Drosophila is quite limited: it can regenerate only imaginal discs but not adult legs or wings, and the body size of Drosophila is too small to be manipulated compared with crickets. Gryllus bimaculatus has remarkable regenerative ability (similarly to the cockroach) and methods to analyze molecular basis of regeneration is established, hence we start to analyze molecular basis of leg regeneration using the cricket.

To study the molecular basis of leg regeneration, we perform RNAi in the third instar nymph and then amputate the metathoracic leg (T3) of the nymph. The crickets grow up to third instar nymphs one week after hatching at 28 degrees. Just after molting from second to third instar, the cuticle of the third instar nymphs is whitish and soft, then turns brownish and blackish, and  hard by a few hours (Figure 1, window). The nymphs are around 5 mm, and look like ants. We collect whitish nymphs into 9 cm petri dish and chill on ice to anesthetize by 30 minutes. To perform RNAi in cricket nymphs, we inject dsRNA into anesthetized cricket nymphs by grass capillary held by a mechanical injector. Injecting is very easy: just insert the capillary into the abdomen of the cricket nymph and push the bottom on the mechanical injector three times (Figure 3). Then we can inject approximately 200 nL of dsRNA solution into the cricket nymph. Two days after performing RNAi, expression of endogenous target gene was decreased.

Each of the cricket prothoracic (T1), mesothoracic (T2) and T3 legs consist of the coxa, trochanter, femur, tibia, tarsus and claws, while the tarsus consists of tarsomere 1 (Ta1), tarsomere 2 (Ta2), and tarsomere 3 (Ta3). To observe regeneration process of the cricket leg, we amputate the legs using ophthalmic scissors. The lost part of the leg is completely restored by the sixth instar (within 2-3 weeks post amputation, Figure 3), and the shape of regenerated legs is indistinguishable from that of intact contralateral legs (Nakamura et al., 2008). Transplantation experiments such as intercalation, reversed intercalation, and tripod formation can be performed on cricket legs (host: T3 leg; graft: T2 leg). In the “tripod experiment”, supernumerary legs formation is induced between the host stump and graft piece (Nakamura et al., 2007) (Figure 3). To transplant a graft piece of T2 leg into the host stump of T3 leg, we just cut T2 leg at the tibia and put the T2 graft piece on the T3 host stump aligning the dorsoventral and anteroposterior axes between host and graft. Since diameters of T3 host stump is bigger than that of T2 graft piece, T2 leg easily fit to T3 leg. There is even no need to use glue to connect the host and graft. After transplantation, the cricket is put on ice 30~60 minutes to anesthetize again, avoiding dislocation of the graft piece from the host stump. The transplanted cricket will molt to next instar nymph after three days, and then newly-formed cuticle continuously covers the host stump and graft piece.

The expression pattern of target genes in the regenerating leg is detectable in the regenerating leg at two, five, or six days post amputation (dpa) by in situ hybridization. Since the cuticles of the insect inhibit penetration of RNA probes, we remove the cuticles from fixed cricket legs. The cuticles of regenerating legs at 2, 5, or 6 dpa are removed using tweezers and micro-scissors under the stereo microscope. The cuticle-removed regenerating legs can be processed for immunostaining using anti-methylated histone H3 lysine 27 (H3K27) (Hamada et al., 2015), anti-5-ethynyl-2′-deoxyuridine (EdU) (Bando et al., 2009), and anti-phosphorylated histone H3 serine 10, and so on (Figure 4).

There are some morphological differences between the T2 and the T3 legs: the T3 leg has three pairs of tibial spur at the distal tibia and one pair of tarsal spur at the distal Ta1. In contrast, the T2 leg has two pairs of tibial spur at the distal tibia and has no tarsal spur at the distal Ta1. Thus, we can select the T3 leg from the T2 leg depending on the purpose of studies. For instance, when I performed RNAi against E(z) and amputation experiments at distal T3 tibia in the third instar nymph, the regenerated leg has an ectopic leg segment between the tibia and tarsus. However, I was not able to determine whether the ectopic segment is tibia or Ta1. If the ectopic segment of T2 regenerated leg has spurs at the end of the segment, we can predict that the segment has a tibia identity. In control crickets, a T2 leg was amputated at a proximal tibia in the third instar nymph, the regenerated leg restored the lost part of tissues including tibia, tibial supers, tarsus and claws. As I expected, in E(z)(RNAi) crickets, the regenerated T2 leg restored the lost part of tissue and the ectopic segment had spurs. These results suggest that the ectopic segment has a tibia identity and morphological character of T2 leg is suitable for regeneration research (Hamada et al., 2015) (Figure 5).

The cricket shows short germ segmentation

A large number of the cricket eggs are collectable at all times of the year. We supply water to the crickets using wet towel paper put into petri dishes. Adult female crickets lay eggs into the wet towel paper. An adult female cricket lays ~3,000 eggs in a few weeks. Usually we keep ~100 adult males and females, hence we can get ~100 eggs within one hour. If we quickly move the towel paper in the water, the laid eggs are removed from the paper, then we can collect the cricket eggs from the water using a tea strainer. We can inject dsRNA for embryonic RNAi, transposons for transgenesis, RNA encoding TALEN or CRISPR/Cas for genome editing, pharmacological reagents and others into the ventroposterior side of the fertilized eggs (Figure 6).

The egg diameter is approximately 3 mm x 0.5 mm. The developmental stage of cricket embryos is well documented from fertilization to hatching. The early embryo is formed on the ventroposterior side of the egg. We dissect fertilized eggs to get cricket embryos in the desired developmental stages (Donoughe and Extavour, 2016). These embryos are applicable for whole mount in situ hybridization to detect gene expression. While the regeneration processes of the leg can be observed during molting, the embryo can be continuously observed and collected anytime for molecular analysis. For efficient RNAi during early embryonic development, we inject dsRNA into the body fluid of adult female (Matsuoka et al., 2015). Early embryos in the ovary intake dsRNA, called parental RNAi. The model animal Drosophila melanogaster shows the long germ segmentation in its developmental mode, while the cricket shows the short germ segmentation in its developmental mode. Thus, in Evo-Devo studies, the cricket is expected to be a more representative model insect.

The cricket shows characteristic circadian rhythm

The cricket shows diurnal activity in nymphal stages, but after imaginal molting, the activity phase is shifted to be nocturnal (Tomioka and Chiba, 1982). Activities of cricket individuals are documented by using small cricket cages with seesaw-like flower, that is a magnetic sensor switch on the flower (Figure 7).

To record the actgram of more smaller animals (the body length of G. bimaculatus is less than 25mm), I have succeeded to utilize LAM25 with an infrared laser sensor for Modicogryllus siamensis (Trikinetics inc). The data from the seesaw and infrared laser locomotor recorder can be analyzed by Actogram J, a plug-in in Image J (unpublished). Control and/or RNAi-treated crickets are reared in the small cage to measure continuous actogram for more than a month. Previously, it was reported that the central clock system is located in the optic lobe (Tomioka and Chiba, 1992), where clock component gene expression is oscillated daily (Tomioka, 2014). In the cricket, the optic lobes locate bilaterally in the brain, at the inside of compound eyes. The clock genes and their expression profiles are similar to those of model animals including mice and flies. For instance, the clock component genes of mice, such as Clock, Bmal1, period1/2/3, Timeless and Cryptochrome are conserved in fly and cricket as Clock (Clk), cycle (cyc), period (per), timeless (tim) and cryptochrome2 (cry2), respectively, and the transcriptional/translational molecular feedback loops for period of 24 hours are consistent between mice and insects. When we measure the activities of per(RNAi) crickets, they show arrhythmic actogram. Furthermore, it is easy to obtain the whole brain, which is also a central clock component, and used for in situ hybridization (Hamada et al., 2009), immunostaining, and so on. Recently, we address the big challenges that elucidate the relationship between leg regeneration and photoperiodism, and systematize knock-in cricket lines using CRISPR/Cas9 system.

The cricket is one of the most familiar insects to enjoy insect songs in autumn in Japan. A Japanese author, Sei Shonagon mentioned that “Evening is the best time in the day in autumn, because we can enjoy the flight of wild geese and songs of insects” in her essay named “Makurano soushi (The Pillow Book)”, written in the 10^th century. Now the cricket is used as a model insect of regeneration biology and RNAi-based analyses show new insights into tissue regeneration in addition to his song. I am thinking about new experiments listening to crickets’ chirp songs today.

References

Bando, T., Mito, T., Maeda, Y., Nakamura, T., Ito, F., Watanabe, T., Ohuchi, H. and Noji, S. (2009). Regulation of leg size and shape by the Dachsous/Fat signalling pathway during regeneration. Development 136, 2235-2245.

Donoughe, S. and Extavour, C. G. (2015). Embryonic development of the cricket Gryllus bimaculatus. Dev Biol 411, 140-156.

Hamada, A., Miyawaki, K., Honda-sumi, E., Tomioka, K., Mito, T., Ohuchi, H. and Noji, S. (2009). Loss-of-function analyses of the fragile X-related and dopamine receptor genes by RNA interference in the cricket Gryllus bimaculatus. Dev Dyn 238, 2025-2033.

Hamada, Y., Bando, T., Nakamura, T., Ishimaru, Y., Mito, T., Noji, S., Tomioka, K. and Ohuchi, H. (2015). Leg regeneration is epigenetically regulated by histone H3K27 methylation in the cricket Gryllus bimaculatus. Development 142, 2916-2927.

Matsuoka, Y., Bando, T., Watanabe, T., Ishimaru, Y., Noji, S., Popadic, A. and Mito, T. (2015). Short germ insects utilize both the ancestral and derived mode of Polycomb group-mediated epigenetic silencing of Hox genes. Biol Open 4, 702-709.

Nakamura, T., Mito, T., Bando, T., Ohuchi, H. and Noji, S. (2008). Dissecting insect leg regeneration through RNA interference. Cell Mol Life Sci 65, 64-72.

Nakamura, T., Mito, T., Tanaka, Y., Bando, T., Ohuchi, H. and Noji, S. (2007). Involvement of canonical Wnt/Wingless signaling in the determination of the positional values within the leg segment of the cricket Gryllus bimaculatus. Dev Growth Differ 49, 79-88.

Nakamura, T., Yoshizaki, M., Ogawa, S., Okamoto, H., Shinmyo, Y., Bando, T., Ohuchi, H., Noji, S. and Mito, T. (2010). Imaging of transgenic cricket embryos reveals cell movements consistent with a syncytial patterning mechanism. Curr Biol 20, 1641-1647.

Tomioka, K. (2014). Chronobiology of crickets: a review. Zoolog Sci 31, 624-632.

Tomioka, K. and Chiba, Y. (1982). Post-embryonic development of circadian rhythm in the cricket, Gryllus bimaculatus: A rhythm reversal. J Comp Physiol 147, 299-304.

Tomioka, K. and Chiba, Y. (1992). Characterization of an optic lobe circadian pacemaker by in situ and in vitro recording of neural activity in the cricket, Gryllus bimaculatus. J Comp Physiol A 171, 1-7.

Watanabe, T., Ochiai, H., Sakuma, T., Horch, H. W., Hamaguchi, N., Nakamura, T., Bando, T., Ohuchi, H., Yamamoto, T., Noji, S., et al. (2012). Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases. Nat Commun 3, 1017.

This post is part of a series on a day in the life of developmental biology labs working on different model organisms. You can read the introduction to the series here and read other posts in this series here.

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This post was originally published on the DMDD blog.

The Zika virus has raised global awareness of birth defects more than at any time in the last 50 years [1]. A recent Nature Editorial explores the opportunities this presents to increase support for vaccination programmes, compulsory fortification of food staples and investment in population-scale databases to mine information on the causes of birth defects. But what does it mean for research into the genetic basis of developmental disorders and rare diseases?

HOW RARE IS RARE?

Some birth defects, such as Zika-linked microcephaly, are infectious in origin. But many are related to genetic mutations, which can also prevent an embryo from developing as it should. The rare diseases resulting from genetic mutations can be chronic and life threatening, and 30% of rare disease patients die before their fifth birthday [2]. Many mutations can also lead to miscarriage. Genetic cardiac conditions affect around 1% of newborn babies [3], but it’s estimated that these defects prevent around ten times as many foetuses from surviving until birth.

A rare disease is difficult to study, because of the relatively small group of patients – by definition less than 1 in 2000 of the general population are affected. But with more than 6000 known rare diseases, 80% of these with a genetic component, it’s likely you or someone you know has a rare disease. 7% of the population will be affected at some point in their lives, which equates to around 3.5 million people in the UK alone [3].

CLUES FROM SYSTEMATIC SCREENS

We have a huge challenge to find and prevent the causes of rare diseases. For those diseases linked to genetic mutations it’s vital that we understand their genetic basis, and basic research in developmental biology is fundamental to this.

Mouse research in particular offers a wealth of information, thanks to systematic studies that would not be possible in human patients. The mouse genome can be manipulated to delete (knock out) a specific gene. The resulting embryos and adult mice can then be studied to look for abnormalities in the way they develop. It’s a unique way to understand the role that a specific gene plays in development from embryo through to adult, and the developmental abnormalities that may arise from a fault with the gene. Around 30% of gene deletions cause abnormalities so severe that the embryo does not survive until birth, so study of these genes also provides clues about the genetic basis of miscarriage.

Systematic screens by the DMDD and IMPC are working to study the effects of individually knocking out every gene in the mouse genome. With all data freely available online, they are a rich resource for those researching human birth defects, miscarriage and developmental disorders.

The Zika virus has put birth defects back on the political agenda, and scientists have a challenge to find and prevent their causes. In this context, systematic screens of mouse gene knockouts and other gene mutations are more important than ever.

DMDD provides a free, online database of embryonic-lethal mouse gene knockouts: see dmdd.org.uk.

REFERENCES

[1] Use Zika to renew focus on birth-defect research, Nature Editorial, Nature 535, 8 (7 July 2016)

[2] About Rare Diseases, Rare Disease UK

[3] Congenital Heart Disease, Centres for Disease Control and Prevention

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Stem cells have the unique properties of both self renewal and differentiation into various lineages. In the last few years, chromatin architecture and noncoding RNAs are emerging as major players in both gene regulation and stem cell differentiation. This November, Abcam is hosting a conference in Sicily to promote this exciting field, and has teamed up with the Node to offer the chance to be the conference’s official Reporter, with free registration!

Are you fascinated by chromatin organization, transcriptional regulation and stem cell biology? Do you want the opportunity to attend a world class meeting in a beautiful setting, to promote your own work and meet leaders in the field? Are you a keen communicator of scientific research?

The Reporter will provide regular updates from the conference via Twitter, and write a meeting report to be published on the Node and Abcam’s website.

Applying is easy: just send a short paragraph (max. 200 words) to events@abcam.com, letting let us know why you think you are the ideal candidate. The winner will receive free registration to the meeting (travel and accommodation not included).

Application deadline: August 1^st

Meeting information

Chairs

Luciano Di Croce & Danny Reinberg

Confirmed speakers

Salvador Aznar Benitah, Shelley Berger, Giacomo Cavalli, Victor Corces, Amanda Fisher, Xiang-Dong Fu, Kristian Helin, Taekyung Kim, Robert Kingston, Tony Kouzarides, Erez Lieberman Aiden, Diego Pasini, Ramin Shiekhattar, Ali Shilatifard, Alexander Tarakhovsky, Maria Elena Torres Padilla and Kenneth Zaret.

 Conference deadlines

Talk abstracts: August 1^st

Poster abstracts: September 8^th

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Comment on “Reprogramming of avian neural crest axial identity and cell fate“, Science 352, 1570-1573, (2016).

Marcos Simoes-Costa, Department of Molecular Biology and Genetics, Cornell University

Marianne Bronner, Division of Biology and Biological Engineering, California Institute of Technology

In the 19^th century, most embryologists (i.e. precursors to developmental biologists) accepted the germ layer theory – the idea that embryos are composed of distinct layers (ectoderm, endoderm and mesoderm), which will give rise to specific tissues and cell types in the adult. For instance, it was believed that all skeletal components of the body were derived from the mesoderm, whereas the ectoderm differentiated into the nervous system and epidermis. Around the turn of the 20^th century, Julia Platt discovered a remarkable exception to this. She used salamander embryos to track neural crest cells, which are an ectodermal progenitor cell population, and observed that they gave rise to skeletal elements of the head (Platt, 1883). These findings were so controversial at the time that Platt was unable to get a faculty position, quit science, and became a politician. But a hundred and twenty years have passed since Platt’s results were published, and time has been kind to her legacy. Today, we know with certainty that the neural crest is indeed an exceptional cell type. During early formation of the central nervous system, neural crest cells delaminate from the neural tube, engage in extensive migration and give rise to a large portion of craniofacial skeleton as well as several other tissues.

This particular question – how ectodermal cells can differentiate into skeletal tissue– captured my imagination and drove my interest toward neural crest biology. Not that this was the only remarkable feature of neural crest cells: they have the ability to undergo epithelial to mesenchymal transition, are migratory, and can to differentiate into multiple cell types. While neural crest cells emerge from the central nervous system along the length of the body axis, only the cranial crest can give rise to the cartilage and bone of the face. This was elegantly demonstrated by Nicole Le Douarin in the 1960s and -70s through the use of chicken-quail chimeras (reviewed in Le Douarin, 1982). Le Douarin and colleagues showed that trunk neural crest cells are unable to form cartilage even if grafted to the head. Strikingly, cranial neural crest grafted to the trunk gave rise to ectopic cartilage nodules, indicating that these cells are autonomously capable of differentiating into mesenchymal tissue. These results demonstrated that the cranial neural crest was intrinsically different from the other crest subpopulations.

Joining Marianne Bronner’s lab at Caltech after finishing my Ph.D. in Brazil, I wanted to learn molecular biology and genomics. The lab is very interested in gene regulatory control of neural crest specification, so my first project, in collaboration with Tatjana Sauka-Spengler and Sonja Mckeown, focused on cis-regulatory control of the transcription factor FoxD3 in chick embryos. FoxD3 is a classic neural crest marker expressed by all neural crest cells along the entire body axis. However, we discovered different enhancers that drove FoxD3 expression in the head versus the trunk: a distal regulatory element (NC2) mediated trunk expression whereas a unique cranial-specific enhancer closer to the FoxD3 promoter (NC1) drove expression in the head. This suggested that axial differences were encoded in the genome (Simoes-Costa et al., 2012), bringing me right back to the question of what makes the cranial neural crest unique. Importantly, our identification of a axial-specific neural crest enhancers provided a new tool for exploring the molecular mechanisms that underlie the development of the cranial crest.

The strategy to investigate the molecular basis of the cranial neural crest’s ability to give rise to cartilage emerged gradually during daily meetings with Marianne, mostly at the coffee machine. During these informal conversations, we delineated an experimental approach to identify factors unique to the cranial crest that drove differentiation into chondrocytes. Marianne had been interested in this question for a long time, and for more personal reasons – while recovering from a bicycle accident (face plant resulting in a broken nose and deviated septum), she learned from the craniofacial surgeon that facial cartilage is very difficult to replace. We decided to employ a combination of comparative transcriptomics, gene regulatory analysis, and classical embryology to identify the molecular players that endow the cranial neural crest with its ability to form cartilage. Crucial to this approach was the fact that the trunk neural crest is unable to make cartilage in vivo. We predicted that by comparing the transcriptomes of the cranial to the trunk populations – that now could be labelled and isolated with the NC1 and NC2 enhancers – we would be able to identify the transcriptional circuitry that activates chondrocytic differentiation in cranial neural crest cells.

Previously, only a couple factors had been identified as being specific to the cranial crest. Id2 was found to be a cranial neural crest gene by a differential display screen performed by Brad Martinsen in Marianne’s lab almost twenty years ago (Martinsen and Bronner-Fraser, 1998). Eric Theveneau and Muriel Altabef also had identified Ets1 as an important player in the development of this cell population (Theveneau et al, 2007). Our comparative transcriptome analysis identified multiple transcription factors that are enriched in the cranial crest, and we proceeded to organize them in a cranial-specific neural crest regulatory circuit. By analyzing their expression patterns during early embryonic development, we defined the hierarchy of the circuit; and by methodically inactivating each gene, we identified the links that integrated these multiple components into a regulatory network.

We were very excited by the identification of this novel cranial-specific regulatory circuit, as it provided important insights about the biology of the cranial neural crest. For instance, it showed that expression of cranial neural crest genes depends upon anterior regulatory information that is laid out during gastrulation. Brn3c, Lhx5, and Dmbx1, which are the components of the first “early” level of the circuit, are all co-expressed adjacent to the anterior neural plate in the early gastrula. These genes are required for the expression of the “later” genes – Sox8, Tfap2b and Ets1 – which are retained in the migratory cranial neural crest. However, one fundamental question remained – could this regulatory circuit be important for the cranial neural crest’s potential to give rise to cartilage and bone?

During our coffee break conversations, Marianne and I came to the conclusion that the best way to tackle this question would be to transplant components of the cranial circuit to trunk cells and see if they would gain the ability to switch fates and differentiate into skeletal tissue. Thus, we decided to revisit the classical experiments of Nicole Le Douarin, but with a twist – we would transfect the trunk neural crest from the donor embryos with components of the cranial circuit before grafting them to the head. We would also trade the quail embryos in favor of transgenic GFP+ chicken embryos, to facilitate tracking of the grafted cells. After surgery, we would let our chimeras grow for a week and verify if the reprogrammed trunk crest differentiated into facial skeleton. At that time, I was eager for a foray into classic cut and paste embryology, thinking that perhaps I would spend a month performing surgeries and a couple of weeks analyzing the chimeric embryos.

Despite my optimism, that was not the case. Grafting is an art and the chimeric embryos are unforgiving of shaky hands. In my daily meetings with Marianne, I was now drinking decaffeinated tea. Since the grafting experiments were taking an enormous amount of time, we realized we needed a more efficient strategy to identify which components of the cranial circuit were able to reprogram the trunk neural crest. And thus, we came up with the idea of using neural crest axial specific enhancers as reporters of cranial identity. The Sox10E2 enhancer from the Sox10 gene, which was identified by Paola Betancur in Marianne’s lab (Betancur et al., 2010), was perfect for this since it is active in migratory cranial neural crest cells and inactive in the trunk. Thus, we proceeded to transfect trunk cells with all different combinations of the cranial circuit components attempting to activate the cranial enhancer in the trunk neural crest.

These experiments lead to the discovery that Sox8, Tfap2b and Ets1 are able turn on the cranial enhancer in the trunk and also to increase expression levels of endogenous cranial genes in these neural crest cells. When the reprogrammed trunk neural crest cells were grafted in the head of a host embryo, they not only gave rise to the normal trunk neural crest cell derivatives, melanocytes and neurons, but these grafts also differentiated into chondrocytes, forming conspicuous cartilage nodules. And thus we had demonstrated that we could use information decoded through gene regulatory studies to reprogram neural crest identity and change its fate in vivo.

I am thrilled with the publication of this paper not only because it represents a great deal of hard work, but also because it answers a long-standing question that has interested me for years. The aspect of the work that I like the most is the marriage between old and new – our results highlight the power of classical embryological approaches, especially when used in conjunction with modern techniques. Moving forward, we will employ these findings to devise novel strategies for in vitro differentiation of facial cartilage. It is our hope that regulatory information derived from embryonic development can inform upon novel protocols for cell and tissue engineering, improving strategies for repair and regeneration.

References:

1. Betancur, P., Bronner-Fraser, M. & Sauka-Spengler, T. Genomic code for Sox10 activation reveals a key regulatory enhancer for cranial neural crest. Proceedings of the National Academy of Sciences of the United States of America 107, 3570-3575, (2010).
2. Le Douarin, N. The neural crest. (Cambridge University Press, 1982).
3. Martinsen, B. J. & Bronner-Fraser, M. Neural crest specification regulated by the helix-loop-helix repressor Id2. Science 281, 988-991, (1998).
4. Platt, J. B. Ectodermic origin of the cartilages of the head. Anat. Anz., 506-509, 1883.
5. Simoes-Costa, M. S., McKeown, S. J., Tan-Cabugao, J., Sauka-Spengler, T. & Bronner, M. E. Dynamic and differential regulation of stem cell factor FoxD3 in the neural crest is Encrypted in the genome. PLoS genetics 8, e1003142, (2012).
6. Simoes-Costa, M., Bronner, M.E. Reprogramming of avian neural crest axial identity and cell fate. Science 352, 1570-1573, (2016).
7. Theveneau, E., Duband, J. L. & Altabef, M. Ets-1 confers cranial features on neural crest delamination. PloS one 2, e1142, (2007).

(44 votes)

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Caroline Dillard and Cédric Maurange

(Aix-Marseille University, CNRS, IBDM, Marseille)

Drosophila neural stem cells as a cancer model

Transformation of a cell into a cancer cell is a complex process along which the cell acquires hallmarks as diverse as resistance to differentiation, infinite proliferative potential, metastasis, resistance to cell death, genome instability (Hanahan and Weinberg, 2011). Moreover, tumours are usually composed of a heterogenous population of cells which may not contribute equally to the tumorigenic process. Indeed, a hierarchy exists among the tumour cells according to their potential to support tumour growth. In order to decipher the mechanisms governing the transformation process, Drosophila has been shown to be a relevant model. In 2005, Caussinus and Gonzalez elegantly demonstrated that impairing neural stem cell asymmetric division in the Drosophila larval brain triggers their amplification and the formation of large neural tumours (Caussinus and Gonzalez, 2005). Moreover, when transplanted into adult Drosophila hosts for long-term in vivo cultivation, the larval brain tumours displayed very quickly several hallmarks of cancer such as unlimited proliferative potential, genome instability or metastatic activity. In our study, we decided to use this model to determine the mechanisms contributing to the malignant transformation. Caussinus and Gonzalez impaired asymmetric neural stem cell division in a large population of neural stem cells in the Drosophila larval central nervous system (CNS). This manipulation leads to the death of the fly before it reaches adulthood. We bypassed this issue by altering only 6 neural stem cells within the larval CNS using a specific promoter. That way the Drosophila is able to survive metamorphosis and enters adulthood. In the CNS of these adults, we observe huge neural stem cell tumours expanding over and over. This observation is particularly interesting given that wild type neural stem cells possess a limited mitotic potential and terminally differentiate at the end of development, being therefore no longer present in the adult CNS. Thus, neural stem cells in tumours acquire another hallmark of cancer during metamorphosis: resistance to differentiation.

Time matters

To terminally differentiate during metamorphosis, the neural stem cell has to journey through a whole developmental program. This program is composed by sequentially expressed transcription factors known as the temporal series. They form an internal clock that governs the identity and the number of neurons generated by each neural stem cell during specific developmental windows. In Drosophila, this neural stem cell-intrinsic clock has been intensively described, particularly, during embryonic and early larval development (Isshiki et al., 2001; Maurange et al., 2008). When the progression in the temporal series is blocked (either by overexpressing or mutating one of the known temporal transcription factors), the neural stem cell stays stuck in this temporal identity and is unable to terminally differentiate during metamorphosis. As a consequence, the neural stem cell persists in the adult CNS. Given that our model of neural stem cell tumour is resistant to differentiation and that temporal series alteration prevents neural stem cells from differentiating during metamorphosis, we wondered whether temporal patterning may be altered in our tumour model.

None of the known temporal transcription factors is relevantly expressed in our tumours at the end of the development demonstrating that the temporal series is not blocked in this context. Nevertheless a target of this early temporal patterning system, the ZBTB transcription factor Chinmo (Maurange et al., 2008), is expressed in a subset of tumour cells suggesting that some cells could have retained an early temporal identity.

To further determine whether developmental time is a crucial component of the neural stem cell malignant transformation, we generated neural stem cell tumours at different moments along development. Tumours were initiated either during early or late larval development. Strikingly, when given the same time to grow, the late-induced tumours were largely unable to resist to terminal differentiation during metamorphosis so that few small benign tumours are found in the adult CNS whereas the early-induced tumours resisted strongly to differentiation and invaded the whole CNS. This observation demonstrates that during early development neural stem cells travel through a window of high malignant susceptibility.

Searching for the mechanisms regulating this early window of malignant susceptibility, we found that the same internal clock that limits the number of neural stem cell divisions also terminates the early window of susceptibility. Indeed, neural stem cells blocked in an early temporal identity remain prone to generate malignant tumours even when the latter are induced during late development. Thus, the temporal series must regulate genes that confer malignant susceptibility.

Malignant neural stem cells stays aberrantly young

Looking at the expression of Chinmo in the early VS late-induced tumours, we observed that Chinmo is largely expressed in the early-induced tumours but absent from the late-induced tumours. Thus, Chinmo expression correlates with malignant susceptibility in the tumorous neural stem cells. In order to establish a causality link between Chinmo’s expression in the neural stem cell and its malignant potential, we conducted gain and loss of function experiments within the early-induced tumours. We were able to show that Chinmo promotes tumour cell proliferation and resistance to differentiation. By means of a transcriptomic analysis, we identified numerous potential targets of Chinmo within the tumours. We selected the two best positive targets of Chinmo as candidates involved in the malignant process: the RNA-binding proteins Imp and Lin-28. Further genetic manipulations within the tumours allowed us to demonstrate that Imp largely contributes to the tumour growth-promoting effect of Chinmo, and Lin-28 at a lesser extent. Interestingly, Imp and Lin-28 exert also a post-transcriptional positive feedback control on Chinmo in the tumour. Thus, Chinmo, Imp and Lin-28 form an oncogenic module supporting the continuous growth of the tumour and its cancerous properties. Interestingly, we could observe that Chinmo, Imp and Lin-28 expression patterns perfectly overlap along normal development. The three genes are co-expressed in young neural stem cells but silenced in older ones. As such they define an early developmental window with specific growth and differentiation-resisting properties. However, in the developmental context, their interdependency is less clear. Together these results suggest that in our tumour model, the sub-population of cells that aberrantly maintains this oncogenic module typical from early development represents the cancer cells that propagate unlimited growth.

Relevance to paediatric cancers in humans

These results therefore demonstrate that Drosophila neural progenitors transit through a window of malignant susceptibility during early development. Moreover they reveal the timing mechanism and oncogenic module that regulate this early malignant susceptibility (Narbonne-Reveau et al., 2016). This is particularly exciting because it may explain why paediatric cancers, that are thought to originate during foetal life in humans, so rapidly become aggressive. Indeed, neural progenitors in mammals are thought to be also temporally patterned along the development although a temporal series has not been clearly identified yet (Naka et al., 2008) (Mattar et al., 2015). Moreover, Imp and Lin-28 are well conserved in mammals where they are expressed during the early development and often present in cancers (Bell et al., 2015; Carmel-Gross et al., 2016; Nielsen et al., 1999; Yang et al., 2015). Further studies would be needed to determine whether and how they promote malignant susceptibility in the early progenitors where they are expressed.

All together, this study helps identify the specific mechanisms that drive the rapid transformation of tumours with early developmental origins. In this context, work on Drosophila continues to illuminate us about the basic principles of life.

Our full paper can be viewed at here.

References

Bell, J.L., Turlapati, R., Liu, T., Schulte, J.H., and Huttelmaier, S. (2015). IGF2BP1 Harbors Prognostic Significance by Gene Gain and Diverse Expression in Neuroblastoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 33, 1285-1293.

Carmel-Gross, I., Bollag, N., Armon, L., and Urbach, A. (2016). LIN28: A Stem Cell Factor with a Key Role in Pediatric Tumor Formation. Stem Cells Dev 25, 367-377.

Caussinus, E., and Gonzalez, C. (2005). Induction of tumor growth by altered stem-cell asymmetric division in Drosophila melanogaster. Nat Genet 37, 1125-1129.

Isshiki, T., Pearson, B., Holbrook, S., and Doe, C.Q. (2001). Drosophila neuroblasts sequentially express transcription factors which specify the temporal identity of their neuronal progeny. Cell 106, 511-521.

Mattar, P., Ericson, J., Blackshaw, S., and Cayouette, M. (2015). A conserved regulatory logic controls temporal identity in mouse neural progenitors. Neuron 85, 497-504.

Maurange, C., Cheng, L., and Gould, A.P. (2008). Temporal transcription factors and their targets schedule the end of neural proliferation in Drosophila. Cell 133, 891-902.

Naka, H., Nakamura, S., Shimazaki, T., and Okano, H. (2008). Requirement for COUP-TFI and II in the temporal specification of neural stem cells in CNS development. Nature neuroscience 11, 1014-1023.

Narbonne-Reveau, K., Lanet, E., Dillard, C., Foppolo, S., Chen, C.H., Parrinello, H., Rialle, S., Sokol, N.S., and Maurange, C. (2016). Neural stem cell-encoded temporal patterning delineates an early window of malignant susceptibility in Drosophila. eLife 5.

Nielsen, J., Christiansen, J., Lykke-Andersen, J., Johnsen, A.H., Wewer, U.M., and Nielsen, F.C. (1999). A family of insulin-like growth factor II mRNA-binding proteins represses translation in late development. Molecular and cellular biology 19, 1262-1270.

Yang, M., Yang, S.L., Herrlinger, S., Liang, C., Dzieciatkowska, M., Hansen, K.C., Desai, R., Nagy, A., Niswander, L., Moss, E.G., et al. (2015). Lin28 promotes the proliferative capacity of neural progenitor cells in brain development. Development 142, 1616-1627.

(3 votes)

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A postdoctoral position is available in the Page laboratory in the Department of Neuroscience at The Scripps Research Institute in Jupiter, Florida to investigate mechanisms of neurogenesis & gliogenesis, axon/dendrite growth and synaptic connectivity in the developing mouse cerebral cortex. A major goal of this project is to understand the influence of genetic risk factors for autism and other neurodevelopmental disorders on these processes at the molecular/cellular, circuit and behavioral levels. For information about the laboratory, please visit: www.scripps.edu/page/.

Requirements:  In addition to a PhD, highly motivated candidates with expertise in developmental biology, genetics, molecular/cell biology, imaging or behavioral neuroscience are encouraged to apply. Interested candidates should send a cover letter, CV and contact details for three references to Damon Page, Ph.D., email: paged@scripps.edu.

TSRI embraces diversity & recognizes it as being a key to our success.  EOE/M/F/V/D

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A fundamental property of all organisms is the remarkable consistency within the formation of the body plan during development. A handful of conserved molecular pathways are responsible for morphological patterning in embryos across different taxa, despite an enormous diversity of embryonic size in nature. Remarkably, morphological patterns are scaled to tissue size at the time of development and same structures are maintained in relative proportions to the overall size.

One classic mechanism of pattern formation is the morphogen model, where a molecule – the morphogen – is secreted from cells at one end of a developmental field, and diffuses into the target tissue (Lander 2007). At any given time point, morphogen concentration is different around cells that are closer versus further away from the morphogen source. The target tissue responds by activating distinct transcriptional programs in progenitor domains that are exposed to different morphogen concentrations, leading to establishment of different cell fates along the length of the tissue. Therefore, size of the target tissue is an integral component of the patterning process.

How is the morphogen mechanism adjusted, then, to trigger the same transcriptional responses in proportional domains across tissues of different sizes in closely related species? In many instances, morphological patterns are set when tissues size is similar: human embryos are roughly the same size during embryogenesis, and differential growth can account for why taller and smaller human individuals nevertheless share similar body proportions. However, in other cases members of related taxa have quite different sizes during embryonic development, when the morphological pattern is being established. How conserved pathways that regulate patterning are modified to generate a size-invariant output is a biological problem that still remains to be solved (Figure 1).

In our recent paper from the Tabin lab, we tackled this problem through the study of aves, a clad of organisms that show a great variation in size – think of the range of egg sizes from an ostrich egg to a hummingbird egg (Uygur et al. 2016). Yet, these animals are related close enough that we would expect patterning mechanisms to be highly conserved. This gave us the flexibility to study how scaling of the body plan is achieved across two organisms with a drastic difference in embryonic size, the chick (Gallus gallus) and the tiny zebra finch (Taeniopygia guttata) (Figure 2). The neural tube provided to be an excellent model system to explore this question in aves. Pioneering studies in the past two decades have shown that the sonic hedgehog (SHH) morphogen secreted from the notochord and floorplate induces distinct transcriptional programs and cell fates at different concentrations along the ventral half of the neural tube (Liem et al. 2000; Dessaud et al. 2007; Stamataki et al. 2005). As is the case with the morphogen model systems, tissue size and distance from source is critical to patterning onset in this context.

When we examined the establishment of pattern in the ventral neural tube of chick versus zebra finch embryos, we were surprised to find out that it is not only the tissue size that varies between the two species, but also the duration of the patterning process. Across highly similar developmental stages and timepoints, the zebra finch ventral neural tube is patterned much faster compared to the chick neural tube. However, when we compare the levels of morphogen secretion, we found out that the zebra finch notochord, which is drastically smaller than the chick notochord, is also generating a morphogen gradient of much lower amplitude throughout development. Even though the smaller species is producing less morphogen, it is patterning the tissue at a much faster rate. What is going on?

In the past, adjustments to the morphogen mediated patterning systems have been explored at the level of adjusting the morphogen gradient itself(Cheung et al. 2014; Gregor et al. 2005; Ben-Zvi & Barkai 2010). Even though this may still be an interesting aspect of the scaling of the neural tube patterning, our findings lead us to explore differences in morphogen responsiveness. According to our initial observations, patterning process in the smaller zebra finch tissue is completed much faster, with less morphogen in the tissue. When exposed to the same concentration of the sonic hedgehog morphogen, would cells from the zebra finch versus chick turn on the same transcriptional program and cell fates? We turned back to classical embryology to answer this question and generated chimeric embryos at a very early time point in development (at 12 hours, to be exact). By transplanting cells between transgenic lines from the two species, we generated embryos with chimeric neural tubes that were made up of both zebra finch and chick cells, where cells from the two species were exposed to the same developmental signals and received the same level of SHH morphogen from a single source. Remarkably, we observed zebra finch cells to be much more sensitive to the morphogen compared to adjacent chick cells that are same distance away from the source. An analysis on the expression of different transcription factors confirmed that the transcriptional profile of the zebra finch cells were of a higher level morphogen threshold, as if they had seen a higher concentration of SHH for longer durations. This shows cell autonomous differences in morphogen sensitivity account for differential response to the morphogen and altered rates of patterning (Figure 3).

The strength of using the neural tube as a model is that the readout for morphogen activity is very well characterized in this system. We took advantage of this to quantify the differential response of neural progenitors to Sonic hedgehog in the two species. In an explant in-vitro culture system, we analyzed response to different concentrations and durations of SHH in neural tube naive explants isolated from either zebra finch or chick embryos. Indeed, all morphogen thresholds that have previously been identified for chick cells were lower for zebra finch cells, meaning that the zebra finch tissue can induce same cell types at lower concentrations and lower exposure durations.

Our studies on the mechanism of this difference showed that the differential response persists even when the SHH pathway is activated directly through its Smoothened receptor via an agonist. This finding revealed that the cell autonomous differential response is an intra-cellular difference downstream of the patched and smoothend receptors. We further showed that differential levels of the sonic hedgehog transcriptional effortors may be responsible for the differential response, as the two species display different levels of the GLI3 transcript. When we manipulated the zebra finch embryos to express higher levels of GLI3, we observed the speed of patterning is altered and the domain ratios are not scaled to the smaller tissue size.

Our study offers a potential explanation to how tissues of different scales achieve proportional domain sizes when using the same signaling pathways for patterning. It appears that in the case of chick and zebra finch, two avian embryos of different sizes, cell intrinsic differences in morphogen sensitivity control scaling of cell fate patterns in the ventral neural tube. We hope this work will provide new venues to study pattern scaling in order to enhance our understanding of how evolutionary mechanisms accommodate adjustments to body size.

Ben-Zvi, D. & Barkai, N., 2010. Scaling of morphogen gradients by an expansion-repression integral feedback control. Proceedings of the National Academy of Sciences of the United States of America, 107(15), pp.6924–9. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2872437&tool=pmcentrez&rendertype=abstract [Accessed July 11, 2014].

Cheung, D. et al., 2014. Adaptation of the length scale and amplitude of the Bicoid gradient profile to achieve robust patterning in abnormally large Drosophila melanogaster embryos. Development (Cambridge, England), 141(1), pp.124–35. Available at: http://www.ncbi.nlm.nih.gov/pubmed/24284208 [Accessed April 12, 2014].

Dessaud, E. et al., 2007. Interpretation of the sonic hedgehog morphogen gradient by a temporal adaptation mechanism. Nature, 450(7170), pp.717–20. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18046410 [Accessed March 21, 2014].

Gregor, T. et al., 2005. Diffusion and scaling during early embryonic pattern formation. Proceedings of the National Academy of Sciences of the United States of America, 102(51), pp.18403–7. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1311912&tool=pmcentrez&rendertype=abstract [Accessed March 26, 2014].

Lander, A.D., 2007. Morpheus unbound: reimagining the morphogen gradient. Cell, 128(2), pp.245–56. Available at: http://www.sciencedirect.com/science/article/pii/S0092867407000517 [Accessed December 10, 2014].

Liem, K.F., Jessell, T.M. & Briscoe, J., 2000. Regulation of the neural patterning activity of sonic hedgehog by secreted BMP inhibitors expressed by notochord and somites. Development (Cambridge, England), 127(22), pp.4855–66. Available at: http://www.ncbi.nlm.nih.gov/pubmed/11044400 [Accessed August 12, 2014].

Stamataki, D. et al., 2005. A gradient of Gli activity mediates graded Sonic Hedgehog signaling in the neural tube. , pp.626–641.

Uygur, A. et al., 2016. Scaling Pattern to Variations in Size during Development of the Vertebrate Neural Tube. Developmental Cell, 37(2), pp.127–135.

(5 votes)

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In this new feature, we conduct a trawl for preprints to find the latest developmental biology and related research from the previous month.

Scientists are increasingly using preprints as a way of releasing research to the community without having to wait for the peer review process, and we want to promote this content here. Several sites host biology preprints, most notably bioRxiv, but also arXiv, f1000 Research, and PeerJ Preprints (for more info on preprints, ASAPbio has a useful FAQ page).

This month we found content from bioRxiv, arXiv, F1000 Research and PeerJ spanning developmental biology research, including investigations into cell fate in preimplantation mouse embryos and worm larvae,  a new technique for bi-allelic gene knockouts, and a method for making midbrain-specific organoids.

Developmental Biology

• Networks that link cytoskeletal regulators and diaphragm proteins underpin filtration function in Drosophila nephrocytes. Simi Muraleedharan, Helen Skaer, Maneesha S Inamdar
• Drosophila sensory cilia lacking MKS-proteins exhibit striking defects during development but only subtle defects in adults. Metta B. Pratt, Joshua S. Titlow, Ilan Davis, Amy R. Barker, Helen R. Dawe, Jordan W. Raff, Helio Roque
• A Facilitated Diffusion Mechanism Establishes the Drosophila Dorsal Gradient. Sophia N Carrell, MIchael D O’Connell, Amy E Allen, Stephanie MSmith, Gregory Reeves
• KIF1A/UNC-104 transports ATG-9 to regulate neurodevelopment and autophagy at synapses. Andrea K.H. Stavoe, Sarah E. Hill, Daniel A. Colón-Ramos
• Antagonistic roles of Polycomb repression and Notch signaling in the maintenance of somatic cell fate in C. elegans. Ringo Pueschel, Francesca Coraggio, Alisha Marti, Peter Meister
• Dynamic changes in Sox2 spatio-temporal expression direct the second cell fate decision through Fgf4/Fgfr2 signaling in preimplantation mouse embryos. Tapan Kumar Mistri, Wibowo Arindrarto, Wei Ping Ng, ChoayangWang, Hiong Lim Leng, Lili Sun, Ian Chambers, ThorstenWohland, Paul Robson
• Kinetochore Microtubules indirectly link Chromosomes and Centrosomes in C. elegans Mitosis. Stefanie Redemann, Johannes Baumgart, Norbert Lindow,Sebastian Fuerthauer, Ehssan Nazockdast, Andrea Kratz, SteffenProhaska, Jan Brugues, Michael Shelley, Thomas Mueller-Reichert
• Probing cytoplasmic viscosity in the confined geometry of tip-growing plant cells via FRAP. James L Kingsley, Jeffrey P Bibeau, Zhilu Chen, Xinming Huang,Luis Vidali, Erkan Tuzel
• RNase reverses segment sequence in the anterior of a beetle egg (Callosobruchus maculatus, Coleoptera). Jitse Michiel van der Meer
• Brachiopods possess a split Hox cluster with signs of spatial, but not temporal collinearity. Sabrina M. Schiemann, Jose M. M. Martin-Duran, Aina Borve,  Bruno C. Vellutini, Yale J. Passamaneck, Andreas Hejnol
• Nanog fluctuations in ES cells highlight the problem of measurement in cell biology. Rosanna C G Smith, Patrick S Stumpf, Sonya J Ridden, AaronSim, Sarah Filippi, Heather Harrington, Ben D MacArthur
• Trace elements during primordial plexiform network formation in human cerebral organoids. Rafaela C Sartore, Simone C Cardoso, Yuri V Lages, Julia M Paraguassu, Rodrigo F Madeiro da Costa, Marilia ZP Guimarães, Carlos A Pérez, Stevens K Rehen

Techniques

• Rapid, one-step generation of biallelic conditional gene knockouts. Bon-Kyoung Koo, Amanda Andersson-Rolf, Roxana C. Mustata,Alessandra Merenda, Sajith Perera, Tiago Grego, Jihoon Kim, KatieAndrews, Juergen Fink, William C. Skarnes
• A novel approach to derive human midbrain-specific organoids from neuroepithelial stem cells. Anna Sophia Monzel, Lisa Maria Smits, Kathrin Hemmer, SihamHachi, Edinson Lucumi Moreno, Thea van Wuellen, Ronan MTFleming, Silvia Bolognin, Jens Christian Schwamborn

Disease modelling

• Zika Virus in the Human Placenta and Developing Brain: Cell Tropism and Drug Inhibition. Hanna Retallack, Elizabeth Di Lullo, Carolina Arias, Kristeene A.Knopp, Carmen Sandoval-Espinosa, Matthew T. Laurie, Yan Zhou,Matthew Gormley, Walter R. Mancia Leon, Robert Krencik, Erik M. Ullian, Julien Spatazza, Alex A. Pollen, Katherine Ona, Tomasz J.Nowakowski, Joseph L. DeRisi, Susan J. Fisher, Arnold R. Kriegstein
• Hipk is required for JAK/STAT activity and promotes hemocyte-derived tumorigenesis. Jessica A. Blaquiere, Nathan B. Wray, Esther M. Verheyen

In silico modelling

• The Evolutionary Trade-off between Stem Cell Niche Size, Aging, and Tumorigenesis.  Vincent L. Cannataro, Scott A. McKinley, Colette M. St. Mary
• Conditions for cell size homeostasis: A stochastic hybrid systems approach. César Augusto Vargas-García, Mohammad Soltani, Abhyudai Singh
• Collective motion of cells crawling on a substrate: roles of cell shape and contact inhibition. Simon Kaspar Schnyder, Yuki Tanaka, John Jairo Molina, Ryoichi Yamamoto

Science Communication

• Not Communicating science? Aiming for national impact. Andreas Prokop and Sam Illingworth

Is there anything we missed for June? Anything in the list that catches your eye? Let us know in the comments section.

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Here are the highlights from the current issue of Development:

A pioneer role for PBX1 in neurogenesis

The adult rodent subventricular zone (SVZ) is a neurogenic niche that provides new neurons and glia to the brain. A number of transcription factors, including MEIS2 and PAX6, are known to be required to promote the neuronal cell fate. PBX family proteins can interact with both MEIS and PAX family factors, and PBX1 is known to be expressed in the adult mouse forebrain. Dorothea Schulte and colleagues therefore set out to test the role of PBX1 in SVZ neurogenesis (p. 2281). They show that Pbx1expression is found in rapidly proliferating SVZ progenitors, as well as in subsets of their progeny in the olfactory bulb. Its ortholog, PBX2, is more widely expressed in the forebrain. Deletion of both genes in the adult SVZ leads to a reduction in neurogenesis and a concomitant increase in oligodendrogliogenesis – an alternative fate for SVZ progenitors. Although the authors can detect PBX1 binding to MEIS2 and PAX6, the phenotype is distinct from functional blockade of these factors, suggesting an at least partially independent function. Intriguingly, PBX1 appears to bind its targets before they are transcriptionally activated, indicating a putative pioneer factor function for PBX1. These data identify PBX1 as an important new regulator of SVZ neurogenesis.

A tyrosine kinase for FERtilisation

Mammalian sperm need to undergo capacitation to become competent to fertilise the oocyte. In vivo, this occurs in the female reproductive tract, whereas in vitro it can be triggered by culture in defined media. One of the key events during capacitation is cAMP-dependent tyrosine phosphorylation of various proteins. Although this phenomenon has been well described in many species, the kinase responsible has been elusive. Various candidates have been proposed, including SRC, FAK and PYK2; notably, inhibitors for the latter two enzymes block tyrosine phosphorylation in human and horse sperm. However, definitive evidence for the identity of the kinase has been lacking. Now (p. 2325), Pablo Visconti and co-workers show that, while FAK and PYK2 inhibitors also block tyrosine phosphorylation in mouse sperm (without blocking PKA activation), neither is the responsible factor. Instead, it is the FER kinase – also targeted by the same inhibitors – that carries out capacitation-associated tyrosine phosphorylation. However, and surprisingly, FER is not required for fertilisation in vivo, suggesting that the presumed involvement of tyrosine phosphorylation in acquisition of sperm competence may need to be revisited.

Coordinating morphogenesis and differentiation with WNT

The mammalian salivary gland is a valuable model for analysis of the morphogenetic and differentiation events that occur during branching morphogenesis of organs. Following a period of growth and branching, epithelial cells of the salivary gland differentiate down ductal or acinar routes. What are the signalling mechanisms that control morphogenesis, differentiation and lineage choice, and how are these processes coordinated to ensure appropriate size and composition of the final organ? The FGF pathway is known to play key roles in salivary gland development, including via the activation of KIT signalling – which promotes the expansion of distal (future acinar) progenitors. Moreover, WNT signalling has been implicated in salivary gland morphogenesis, although its function is unclear. On p.2311, Akira Kikuchi and colleagues use genetic and pharmacological approaches, both in vivo and in vitro, to demonstrate that mesenchymally derived WNT signals inhibit acinar differentiation and maintain end bud cells in an undifferentiated state, to promote secondary and tertiary duct formation. Mechanistically, WNT activity promotes MYB-dependent inhibition of Kit expression; KIT directs acinar differentiation through AKT. As WNT pathway activity is dynamically regulated, the authors propose this as a means by which salivary gland morphogenesis and differentiation can be spatiotemporally coordinated.

PLUS!

Cell behaviors and dynamics during angiogenesis

Vascular networks are formed and maintained through a multitude of angiogenic processes, such as sprouting, anastomosis and pruning. Only recently has it become possible to study the behavior of the endothelial cells that contribute to these networks at a single-cell level in vivo. Here, Markus Affolter and colleagues summarizes what is known about endothelial cell behavior during developmental angiogenesis, focusing on the morphogenetic changes that these cells undergo. See the Review on. p. 2249.

Generation of intestinal surface: an absorbing tale

The vertebrate small intestine requires an enormous surface area to effectively absorb nutrients from food. Morphological adaptations required to establish this extensive surface include generation of an extremely long tube and convolution of the absorptive surface of the tube into villi and microvilli. In their Review, Deborah Gumucio and colleagues discuss recent findings regarding the morphogenetic and molecular processes required for intestinal tube elongation and surface convolution in different species. See the Review on p. 2261.

Dental mesenchymal stem cells

Mammalian teeth harbour mesenchymal stem cells (MSCs), which contribute to tooth growth and repair. These dental MSCs possess many in vitro features of bone marrow-derived MSCs, including clonogenicity, expression of certain markers, and following stimulation, differentiation into cells that have the characteristics of osteoblasts, chondrocytes and adipocytes. Here, Paul Sharpe outlines some recent discoveries in dental MSC function and behaviour and discusses how these and other advances are paving the way for the development of new biologically based dental therapies. See the Review on p. 2273.

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