Author Correction: Elephant shark genome provides unique insights into gnathostome evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Signalling change: signal transduction through the decades.

The past few years have marked significant anniversaries in signal transduction, including the identification of classic growth factors and morphogens, the notion of protein modification through phosphorylation and the characterization of protein interaction domains. Here, six researchers reflect on the context in which these discoveries were made, and how our concept of cell signalling has evolved during the past three decades.

Blood vessel occlusion by Cryptococcus neoformans is a mechanism for haemorrhagic dissemination of infection.

Meningitis caused by infectious pathogens is associated with vessel damage and infarct formation, however the physiological cause is often unknown. Cryptococcus neoformans is a human fungal pathogen and causative agent of cryptococcal meningitis, where vascular events are observed in up to 30% of patients, predominantly in severe infection. Therefore, we aimed to investigate how infection may lead to vessel damage and associated pathogen dissemination using a zebrafish model that permitted noninvasive in vivo imaging. We find that cryptococcal cells become trapped within the vasculature (dependent on their size) and proliferate there resulting in vasodilation. Localised cryptococcal growth, originating from a small number of cryptococcal cells in the vasculature was associated with sites of dissemination and simultaneously with loss of blood vessel integrity. Using a cell-cell junction tension reporter we identified dissemination from intact blood vessels and where vessel rupture occurred. Finally, we manipulated blood vessel tension via cell junctions and found increased tension resulted in increased dissemination. Our data suggest that global vascular vasodilation occurs following infection, resulting in increased vessel tension which subsequently increases dissemination events, representing a positive feedback loop. Thus, we identify a mechanism for blood vessel damage during cryptococcal infection that may represent a cause of vascular damage and cortical infarction during cryptococcal meningitis.

Development of the electric organ in embryos and larvae of the knifefish, Brachyhypopomus gauderio.

South American Gymnotiform knifefish possess electric organs that generate electric fields for electro-location and electro-communication. Electric organs in fish can be derived from either myogenic cells (myogenic electric organ/mEO) or neurogenic cells (neurogenic electric organ/nEO). To date, the embryonic development of EOs has remained obscure. Here we characterize the development of the mEO in the Gymnotiform bluntnose knifefish, Brachyhypopomus gauderio. We find that EO primordial cells arise during embryonic stages in the ventral edge of the tail myotome, translocate into the ventral fin and develop into syncytial electrocytes at early larval stages. We also describe a pair of thick nerve cords that flank the dorsal aorta, the location and characteristic morphology of which are reminiscent of the nEO in Apteronotid species, suggesting a common evolutionary origin of these tissues. Taken together, our findings reveal the embryonic origins of the mEO and provide a basis for elucidating the mechanisms of evolutionary diversification of electric charge generation by myogenic and neurogenic EOs.

From Drosophila segmentation to human cancer therapy.

First described in Drosophila, Hedgehog signalling is a key regulator of embryonic development and tissue homeostasis and its dysfunction underlies a variety of human congenital anomalies and diseases. Although now recognised as a major target for cancer therapy as well as a mediator of directed stem cell differentiation, the unveiling of the function and mechanisms of Hedgehog signalling was driven largely by an interest in basic developmental biology rather than clinical need. Here, I describe how curiosity about embryonic patterning led to the identification of the family of Hedgehog signalling proteins and the pathway that transduces their activity, and ultimately to the development of drugs that block this pathway.

Elephant shark genome provides unique insights into gnathostome evolution.

The emergence of jawed vertebrates (gnathostomes) from jawless vertebrates was accompanied by major morphological and physiological innovations, such as hinged jaws, paired fins and immunoglobulin-based adaptive immunity. Gnathostomes subsequently diverged into two groups, the cartilaginous fishes and the bony vertebrates. Here we report the whole-genome analysis of a cartilaginous fish, the elephant shark (Callorhinchus milii). We find that the C. milii genome is the slowest evolving of all known vertebrates, including the 'living fossil' coelacanth, and features extensive synteny conservation with tetrapod genomes, making it a good model for comparative analyses of gnathostome genomes. Our functional studies suggest that the lack of genes encoding secreted calcium-binding phosphoproteins in cartilaginous fishes explains the absence of bone in their endoskeleton. Furthermore, the adaptive immune system of cartilaginous fishes is unusual: it lacks the canonical CD4 co-receptor and most transcription factors, cytokines and cytokine receptors related to the CD4 lineage, despite the presence of polymorphic major histocompatibility complex class II molecules. It thus presents a new model for understanding the origin of adaptive immunity.

Open questions: how to get developmental biology into shape?

Recent technical advances have provided unprecedented insights into the selective deployment of the genome in developing organisms, but how such differential gene expression is used to sculpt the complex shapes and sizes of organs remains unclear. Here, we outline major open questions in organogenesis and suggest how a synthesis between developmental biology and physics can help to address them.

Zebrafish genetics gets the Scube on Hedgehog secretion.

Inspired by a zebrafish mutation, two recent studies by Creanga and colleagues (pp. 1312-1325) and Tukachinsky and colleagues have shed new light on the way in which lipidated Hedgehog proteins are secreted and released from expressing cells, suggesting a model for the sequential action of the Disp and Scube2 proteins in this process.

Hedgehog signalling.

The Hedgehog (Hh) signalling pathway is one of the key regulators of metazoan development. Hh proteins have been shown to play roles in many developmental processes and have become paradigms for classical morphogens. Dysfunction of the Hh pathway underlies a number of human developmental abnormalities and diseases, making it an important therapeutic target. Interest in Hh signalling thus extends across many fields, from evo-devo to cancer research and regenerative medicine. Here, and in the accompanying poster, we provide an outline of the current understanding of Hh signalling mechanisms, highlighting the similarities and differences between species.

The transcription factor SOX6 contributes to the developmental origins of obesity by promoting adipogenesis.

An association between impaired fetal growth and the postnatal development of obesity has been established. Here, by comparing adipocytes differentiated from mesenchymal stem cells (MSCs) taken from the umbilical cord and derived from normal and growth-restricted neonates, we identified the transcription factor SOX6 as highly expressed only in growth-restricted individuals. We found that SOX6 regulates adipogenesis in vertebrate species by activating adipogenic regulators including PPARγ, C/EBPα and MEST. We further show that SOX6 interacts with ß-catenin in adipocytes, suggesting an inhibition of WNT/ß-catenin signaling, thereby promoting adipogenesis. The upstream regulatory region of the MEST gene in MSCs from growth-restricted subjects harbors hypomethylated CpGs next to SOX6 binding motifs, and we found that SOX6 binding is impaired by adjacent CpG methylation. In summary, we report that SOX6 is a novel regulator of adipogenesis synergizing with epigenetic mechanisms.

Engrailed controls epaxial-hypaxial muscle innervation and the establishment of vertebrate three-dimensional mobility.

Chordates are characterised by contractile muscle on either side of the body that promotes movement by side-to-side undulation. In the lineage leading to modern jawed vertebrates (crown group gnathostomes), this system was refined: body muscle became segregated into distinct dorsal (epaxial) and ventral (hypaxial) components that are separately innervated by the medial and hypaxial motors column, respectively, via the dorsal and ventral ramus of the spinal nerves. This allows full three-dimensional mobility, which in turn was a key factor in their evolutionary success. How the new gnathostome system is established during embryogenesis and how it may have evolved in the ancestors of modern vertebrates is not known. Vertebrate Engrailed genes have a peculiar expression pattern as they temporarily demarcate a central domain of the developing musculature at the epaxial-hypaxial boundary. Moreover, they are the only genes known with this particular expression pattern. The aim of this study was to investigate whether Engrailed genes control epaxial-hypaxial muscle development and innervation. Investigating chick, mouse and zebrafish as major gnathostome model organisms, we found that the Engrailed expression domain was associated with the establishment of the epaxial-hypaxial boundary of muscle in all three species. Moreover, the outgrowing epaxial and hypaxial nerves orientated themselves with respect to this Engrailed domain. In the chicken, loss and gain of Engrailed function changed epaxial-hypaxial somite patterning. Importantly, in all animals studied, loss and gain of Engrailed function severely disrupted the pathfinding of the spinal motor axons, suggesting that Engrailed plays an evolutionarily conserved role in the separate innervation of vertebrate epaxial-hypaxial muscle.

An essential role for Grk2 in Hedgehog signalling downstream of Smoothened.

The G-protein-coupled receptor kinase 2 (adrbk2/GRK2) has been implicated in vertebrate Hedgehog (Hh) signalling based on the effects of its transient knock-down in mammalian cells and zebrafish embryos. Here, we show that the response to Hh signalling is effectively abolished in the absence of Grk2 activity. Zebrafish embryos lacking all Grk2 activity are refractory to both Sonic hedgehog (Shh) and oncogenic Smoothened (Smo) activity, but remain responsive to inhibition of cAMP-dependent protein kinase (PKA) activity. Mutation of the kinase domain abrogates the rescuing activity of grk2 mRNA, suggesting that Grk2 acts in a kinase-dependent manner to regulate the response to Hh. Previous studies have suggested that Grk2 potentiates Smo activity by phosphorylating its C-terminal tail (CTT). In the zebrafish embryo, however, phosphomimetic Smo does not display constitutive activity, whereas phospho-null mutants retain activity, implying phosphorylation is neither sufficient nor necessary for Smo function. Since Grk2 rescuing activity requires the integrity of domains essential for its interaction with GPCRs, we speculate that Grk2 may regulate Hh pathway activity by downregulation of a GPCR.

Divergence of zebrafish and mouse lymphatic cell fate specification pathways.

In mammals, the homeodomain transcription factor Prox1 acts as the central regulator of lymphatic cell fate. Its restricted expression in a subset of cardinal vein cells leads to a switch towards lymphatic specification and hence represents a prerequisite for the initiation of lymphangiogenesis. Murine Prox1-null embryos lack lymphatic structures, and sustained expression of Prox1 is indispensable for the maintenance of lymphatic cell fate even at adult stages, highlighting the unique importance of this gene for the lymphatic lineage. Whether this pre-eminent role of Prox1 within the lymphatic vasculature is conserved in other vertebrate classes has remained unresolved, mainly owing to the lack of availability of loss-of-function mutants. Here, we re-examine the role of Prox1a in zebrafish lymphangiogenesis. First, using a transgenic reporter line, we show that prox1a is initially expressed in different endothelial compartments, becoming restricted to lymphatic endothelial cells only at later stages. Second, using targeted mutagenesis, we show that Prox1a is dispensable for lymphatic specification and subsequent lymphangiogenesis in zebrafish. In line with this result, we found that the functionally related transcription factors Coup-TFII and Sox18 are also dispensable for lymphangiogenesis. Together, these findings suggest that lymphatic commitment in zebrafish and mice is controlled in fundamentally different ways.

Mechanisms and functions of Hedgehog signalling across the metazoa.

Hedgehog proteins constitute one of a small number of families of secreted signals that have a central role in the development of metazoans. Genetic analyses in flies, fish and mice have uncovered the major components of the pathway that transduces Hedgehog signals, and recent genome sequence projects have provided clues about its evolutionary origins. In this Review we provide an updated overview of the mechanisms and functions of this signalling pathway, highlighting the conserved and divergent features of the pathway, as well as some of the common themes in its deployment that have emerged from recent studies.

Serum and glucocorticoid-regulated kinase 1 regulates neutrophil clearance during inflammation resolution.

The inflammatory response is integral to maintaining health by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralize invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein serum and glucocorticoid-regulated kinase 1 (SGK1). We have characterized the expression patterns and regulation of SGK family members in human neutrophils and shown that inhibition of SGK activity completely abrogates the antiapoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signaling and thus may prove a valuable therapeutic target for the treatment of inflammatory disease.

Adaxial cell migration in the zebrafish embryo is an active cell autonomous property that requires the Prdm1a transcription factor.

Adaxial cells, the progenitors of slow-twitch muscle fibres in zebrafish, exhibit a stereotypic migratory behaviour during somitogenesis. Although this process is known to be disrupted in various mutants, its precise nature has remained unclear. Here, using in vivo imaging and chimera analysis, we show that adaxial cell migration is a cell autonomous process, during which cells become polarised and extend filopodia at their leading edge. Loss of function of the Prdm1a transcription factor disrupts the polarisation and migration of adaxial cells, reflecting a role that is independent of its repression of sox6 expression. Expression of the M- and N-cadherins, previously implicated in driving adaxial cell migration, is largely unaffected by loss of Prdm1a function, suggesting that differential cadherin expression is not sufficient for adaxial cell migration.

Deep sequencing of small RNA facilitates tissue and sex associated microRNA discovery in zebrafish.

BACKGROUND: The role of microRNAs in gene regulation has been well established. The extent of miRNA regulation also increases with increasing genome complexity. Though the number of genes appear to be equal between human and zebrafish, substantially less microRNAs have been discovered in zebrafish compared to human (miRBase Release 19). It appears that most of the miRNAs in zebrafish are yet to be discovered. RESULTS: We sequenced small RNAs from brain, gut, liver, ovary, testis, eye, heart and embryo of zebrafish. In brain, gut and liver sequencing was done sex specifically. Majority of the sequenced reads (16-62 %) mapped to known miRNAs, with the exception of ovary (5.7 %) and testis (7.8 %). Using the miRNA discovery tool (miRDeep2), we discovered novel miRNAs from the unannotated reads that ranged from 7.6 to 23.0 %, with exceptions of ovary (51.4 %) and testis (55.2 %). The prediction tool identified a total of 459 novel pre-miRNAs. We compared expression of miRNAs between different tissues and between males and females to identify tissue associated and sex associated miRNAs respectively. These miRNAs could serve as putative biomarkers for these tissues. The brain and liver had highest number of tissue associated (22) and sex associated (34) miRNAs, respectively. CONCLUSIONS: This study comprehensively identifies tissue and sex associated miRNAs in zebrafish. Further, we have discovered 459 novel pre-miRNAs (~30 % seed homology to human miRNA) as a genomic resource which can facilitate further investigations to understand miRNA-mRNA gene regulatory networks in zebrafish which will have implications in understanding the function of human homologs.

Tardbpl splicing rescues motor neuron and axonal development in a mutant tardbp zebrafish.

Mutations in the transactive response DNA binding protein-43 (TARDBP/TDP-43) gene, which regulates transcription and splicing, causes a familial form of amyotrophic lateral sclerosis (ALS). Here, we characterize and report the first tardbp mutation in zebrafish, which introduces a premature stop codon (Y220X), eliminating expression of the Tardbp protein. Another TARDBP ortholog, tardbpl, in zebrafish is shown to encode a Tardbp-like protein which is truncated compared with Tardbp itself and lacks part of the C-terminal glycine-rich domain (GRD). Here, we show that tardbp mutation leads to the generation of a novel tardbpl splice form (tardbpl-FL) capable of making a full-length Tardbp protein (Tardbpl-FL), which compensates for the loss of Tardbp. This finding provides a novel in vivo model to study TDP-43-mediated splicing regulation. Additionally, we show that elimination of both zebrafish TARDBP orthologs results in a severe motor phenotype with shortened motor axons, locomotion defects and death at around 10 days post fertilization. The Tardbp/Tardbpl knockout model generated in this study provides an excellent in vivo system to study the role of the functional loss of Tardbp and its involvement in ALS pathogenesis.