Prof Tatjana Sauka-Spengler

Research Area: Genetics and Genomics
Technology Exchange: Bioinformatics, Cell sorting, Computational biology, ES cell / homologous recombination, Immunohistochemistry, In situ hybridisation, In vivo imaging, Mass spectrometry, Microscopy (Confocal), Protein interaction, Transcript profiling and Transgenesis
Scientific Themes: Genes, Genetics, Epigenetics & Genomics and Developmental Biology & Stem Cells
Keywords: gene regulatory networks, cell type-specific epigenomic and transcriptional profiling, neural crest
Web Links:
Cranial neural crest in chick embryo expressing Foxd3 (green), Sox10 (red) and Msx1 (blue) transcription factors

Cranial neural crest in chick embryo expressing Foxd3 (green), Sox10 (red) and Msx1 (blue) ...

Enhancer activity in neural crest cells in the chick embryos (FoxD3 in red, Sox10 in green)

Enhancer activity in neural crest cells in the chick embryos (FoxD3 in red, Sox10 in green)

Zebrafish binary in vivo biotinylation system enables tissue-specific biotin labelling of Avi-tagged proteins and consists of two types of transgenic lines: (i) tissue-specific driver lines, expressing BirA (example: left panels, Sox10-BirA-membCherry) and (ii) effector lines, expressing Avi-tagged version of protein of interest (example: right panels, outer nuclear envelope-tethered tagged protein, RanGap-Cerulean-Avi)

Zebrafish binary in vivo biotinylation system enables tissue-specific biotin labelling of ...

Confocal image of embryo obtained by crossing ubiquitous effector Avi Cerulean RanGap zebrafish line (in red) to neural crest-specific BirA driver line (in green). Biotinylated neural crest nuclei (red nuclear envelope in green cells) can be easily purified from the total nuclear pool and subjected to epigenomic or transcriptional profiling.

Confocal image of embryo obtained by crossing ubiquitous effector Avi Cerulean RanGap zebrafish ...

Embryonic development is driven by a large set of finely orchestrated regulatory programs that control cell fate decisions, differentiation and morphogenesis, leading to formation of a complex organism. Understanding how these programs, encoded at the genome level, are translated into intricate networks of interacting biological components (genes, proteins, RNA) is essential to our understanding of mechanisms underlying developmental processes and human diseases, triggered when biological circuits go awry.

Modern epigenomic techniques are powerful tools to dissect complex regulatory networks, providing the ability to systematically analyse chromatin landscape and on-going transcriptional programs. Epigenomic profiling of histone modifications allows genome-wide chromatin signature mapping and classification of sites of regulatory activity (e.g. distal elements/enhancers, promoters, repressed regions, etc.). Concomitantly, active transcriptome analysis provides information about upstream inputs and downstream outputs within the Gene Regulatory Networks (GRNs) that orchestrate diverse cellular processes.

In order to mechanistically dissect GRNs during development, we are adapting systems level approaches, such as epigenomic and transcriptional profiling, to defined cell populations in the developing embryo.  The data sets obtained allow us to annotate the sites of regulatory activity, and consequently assemble and test gene regulatory circuitry that controls given developmental process at the cellular level. We use two developmental models, the chicken (1) and the zebrafish (2) embryo.

(1) One of the main efforts in our laboratory is building a systems level understanding of the gene regulatory network that orchestrates early steps of neural crest formation in vertebrate embryos. We use the chicken embryo, a classical model for studying neural crest, whose mode of early development closely resembles human. The neural crest is multipotent, embryonic stem cell-like population which gives rise to a plethora of derivative tissues and organs, such as sensory and autonomic ganglia, adrenal and thyroid glands, smooth muscle of major blood vessels, craniofacial skeleton and the vast majority of body’s pigmentation. Due to their unique multipotency, coupled with the developmental plasticity, there is broad interest in using the regenerative capacity of neural crest cells in stem cell-based treatments. By deciphering GRNs that orchestrate early steps of neural crest formation, we aim to understand the mechanistic basis of their multipotency and stem-cell like potential, as well as and the biochemical hierarchy that controls the maintenance of those properties.

(2) In addition to being a powerful organism for studying embryonic development, in recent years zebrafish has become an important system for biomedical research, as one of the keys to understanding human disease and addressing critical questions in regenerative medicine. Zebrafish not only have the same genes as humans but also most of the same cell types, tissues, organs and biological circuits, assuring that the lessons from this model can be directly applied to other vertebrate systems and to human health.

We have developed a versatile, genetically encoded, binary in vivo biotinylation approach in zebrafish, which allows for tissue-specific biotinylation of defined targets. This is achieved by co-expression of proteins tagged with biotin acceptor peptide (Avi-tag) and bacterial biotin ligase, BirA in the same cells, allowing to isolate specific proteins or cell populations, using single step affinity purification procedure.  Isolated genetically defined cell populations can then be profiled using genome-wide assays, adapted to small cell numbers. We are using this technology to analyze cellular circuitry at as many levels as possible and to address transcriptional and epigenomic mechanisms at play in an array of developmental systems, such as neural crest and hematopoietic lineages, but also in processes activated during inflammatory response to injury and cancer, or during organ regeneration.

Name Department Institution Country
Prof Marianne Bronner California Institute of Technology United States
Prof Scott Fraser California Institute of Technology United States
Prof Niles Pierce California Institute of Technology United States
Prof Vincenzo Cerundolo Investigative Medicine Division Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Prof Roger Patient Nuffield Division of Clinical Laboratory Sciences Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Prof Andrea Streit Craniofacial Development & Orthodontics King's College London United Kingdom
Prof Anna L Gloyn OCDEM Oxford University, Oxford Centre for Diabetes, Endocrinology & Metabolism United Kingdom
Prof Paul Riley DPAG University of Oxford United Kingdom
Choi HM, Calvert CR, Husain N, Huss D, Barsi JC, Deverman BE, Hunter RC, Kato M, Lee SM, Abelin AC et al. 2016. Mapping a multiplexed zoo of mRNA expression. Development, 143 (19), pp. 3632-3637. | Show Abstract | Read more

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences.

Hay D, Hughes JR, Babbs C, Davies JO, Graham BJ, Hanssen LL, Kassouf MT, Oudelaar AM, Sharpe JA, Suciu MC et al. 2016. Genetic dissection of the α-globin super-enhancer in vivo. Nat Genet, 48 (8), pp. 895-903. | Show Abstract | Read more

Many genes determining cell identity are regulated by clusters of Mediator-bound enhancer elements collectively referred to as super-enhancers. These super-enhancers have been proposed to manifest higher-order properties important in development and disease. Here we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer individually and in informative combinations, we demonstrate that each constituent enhancer seems to act independently and in an additive fashion with respect to hematological phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects. Our study highlights the importance of functional genetic analyses for the identification of new concepts in transcriptional regulation.

Hellner K, Miranda F, Fotso Chedom D, Herrero-Gonzalez S, Hayden DM, Tearle R, Artibani M, KaramiNejadRanjbar M, Williams R, Gaitskell K et al. 2016. Premalignant SOX2 overexpression in the fallopian tubes of ovarian cancer patients: Discovery and validation studies. EBioMedicine, 10 pp. 137-149. | Show Abstract | Read more

Current screening methods for ovarian cancer can only detect advanced disease. Earlier detection has proved difficult because the molecular precursors involved in the natural history of the disease are unknown. To identify early driver mutations in ovarian cancer cells, we used dense whole genome sequencing of micrometastases and microscopic residual disease collected at three time points over three years from a single patient during treatment for high-grade serous ovarian cancer (HGSOC). The functional and clinical significance of the identified mutations was examined using a combination of population-based whole genome sequencing, targeted deep sequencing, multi-center analysis of protein expression, loss of function experiments in an in-vivo reporter assay and mammalian models, and gain of function experiments in primary cultured fallopian tube epithelial (FTE) cells. We identified frequent mutations involving a 40kb distal repressor region for the key stem cell differentiation gene SOX2. In the apparently normal FTE, the region was also mutated. This was associated with a profound increase in SOX2 expression (p<2(-16)), which was not found in patients without cancer (n=108). Importantly, we show that SOX2 overexpression in FTE is nearly ubiquitous in patients with HGSOCs (n=100), and common in BRCA1-BRCA2 mutation carriers (n=71) who underwent prophylactic salpingo-oophorectomy. We propose that the finding of SOX2 overexpression in FTE could be exploited to develop biomarkers for detecting disease at a premalignant stage, which would reduce mortality from this devastating disease.

Bogdanović O, Smits AH, de la Calle Mustienes E, Tena JJ, Ford E, Williams R, Senanayake U, Schultz MD, Hontelez S, van Kruijsbergen I et al. 2016. Active DNA demethylation at enhancers during the vertebrate phylotypic period. Nat Genet, 48 (4), pp. 417-426. | Show Abstract | Read more

The vertebrate body plan and organs are shaped during a conserved embryonic phase called the phylotypic stage. However, the mechanisms that guide the epigenome through this transition and their evolutionary conservation remain elusive. Here we report widespread DNA demethylation of enhancers during the phylotypic period in zebrafish, Xenopus tropicalis and mouse. These enhancers are linked to developmental genes that display coordinated transcriptional and epigenomic changes in the diverse vertebrates during embryogenesis. Binding of Tet proteins to (hydroxy)methylated DNA and enrichment of 5-hydroxymethylcytosine in these regions implicated active DNA demethylation in this process. Furthermore, loss of function of Tet1, Tet2 and Tet3 in zebrafish reduced chromatin accessibility and increased methylation levels specifically at these enhancers, indicative of DNA methylation being an upstream regulator of phylotypic enhancer function. Overall, our study highlights a regulatory module associated with the most conserved phase of vertebrate embryogenesis and suggests an ancient developmental role for Tet dioxygenases.

Gu W, Monteiro R, Zuo J, Simões FC, Martella A, Andrieu-Soler C, Grosveld F, Sauka-Spengler T, Patient R. 2015. A novel TGFβ modulator that uncouples R-Smad/I-Smad-mediated negative feedback from R-Smad/ligand-driven positive feedback. PLoS Biol, 13 (2), pp. e1002051. | Show Abstract | Read more

As some of the most widely utilised intercellular signalling molecules, transforming growth factor β (TGFβ) superfamily members play critical roles in normal development and become disrupted in human disease. Establishing appropriate levels of TGFβ signalling involves positive and negative feedback, which are coupled and driven by the same signal transduction components (R-Smad transcription factor complexes), but whether and how the regulation of the two can be distinguished are unknown. Genome-wide comparison of published ChIP-seq datasets suggests that LIM domain binding proteins (Ldbs) co-localise with R-Smads at a substantial subset of R-Smad target genes including the locus of inhibitory Smad7 (I-Smad7), which mediates negative feedback for TGFβ signalling. We present evidence suggesting that zebrafish Ldb2a binds and directly activates the I-Smad7 gene, whereas it binds and represses the ligand gene, Squint (Sqt), which drives positive feedback. Thus, the fine tuning of TGFβ signalling derives from positive and negative control by Ldb2a. Expression of ldb2a is itself activated by TGFβ signals, suggesting potential feed-forward loops that might delay the negative input of Ldb2a to the positive feedback, as well as the positive input of Ldb2a to the negative feedback. In this way, precise gene expression control by Ldb2a enables an initial build-up of signalling via a fully active positive feedback in the absence of buffering by the negative feedback. In Ldb2a-deficient zebrafish embryos, homeostasis of TGFβ signalling is perturbed and signalling is stably enhanced, giving rise to excess mesoderm and endoderm, an effect that can be rescued by reducing signalling by the TGFβ family members, Nodal and BMP. Thus, Ldb2a is critical to the homeostatic control of TGFβ signalling and thereby embryonic patterning.

Uy BR, Simoes-Costa M, Koo DE, Sauka-Spengler T, Bronner ME. 2015. Evolutionarily conserved role for SoxC genes in neural crest specification and neuronal differentiation. Dev Biol, 397 (2), pp. 282-292. | Show Abstract | Read more

Members of the Sox family of transcription factors play a variety of critical developmental roles in both vertebrates and invertebrates. Whereas SoxBs and SoxEs are involved in neural and neural crest development, respectively, far less is known about members of the SoxC subfamily. To address this from an evolutionary perspective, we compare expression and function of SoxC genes in neural crest cells and their derivatives in lamprey (Petromyzon marinus), a basal vertebrate, to frog (Xenopus laevis). Analysis of transcript distribution reveals conservation of lamprey and X. laevis SoxC expression in premigratory neural crest, branchial arches, and cranial ganglia. Moreover, morpholino-mediated loss-of-function of selected SoxC family members demonstrates essential roles in aspects of neural crest development in both organisms. The results suggest important and conserved functions of SoxC genes during vertebrate evolution and a particularly critical, previously unrecognized role in early neural crest specification.

Betancur P, Simões-Costa M, Sauka-Spengler T, Bronner ME. 2014. Expression and function of transcription factor cMyb during cranial neural crest development. Mech Dev, 132 pp. 38-43. | Show Abstract | Read more

The transcription factor cMyb has well known functions in vertebrate hematopoiesis, but little was known about its distribution or function at early developmental stages. Here, we show that cMyb transcripts are present at the neural plate during gastrulation in chick embryos. cMyb expression then resolves to the cranial neural folds and is maintained in early migrating cranial neural crest cells during and after neurulation. Morpholino-mediated knock-down of cMyb reduces expression of Pax7 and Twist at the neural plate border, as well as reducing expression of neural crest specifier gene Slug/Snail2 and completely eliminating expression of Ets1. On the other hand, its loss results in abnormal maintenance of Zic1, but little or no effect on other neural crest specifier genes like FoxD3 or Sox9. These results place cMyb in a critical hierarchical position within the cranial neural crest cell gene regulatory network, likely directly inhibiting Zic1 and upstream of Ets1 and some, but not all, neural crest specifier genes.

Modrell MS, Hockman D, Uy B, Buckley D, Sauka-Spengler T, Bronner ME, Baker CVH. 2014. A fate-map for cranial sensory ganglia in the sea lamprey Developmental Biology, 385 (2), pp. 405-416. | Show Abstract | Read more

Cranial neurogenic placodes and the neural crest make essential contributions to key adult characteristics of all vertebrates, including the paired peripheral sense organs and craniofacial skeleton. Neurogenic placode development has been extensively characterized in representative jawed vertebrates (gnathostomes) but not in jawless fishes (agnathans). Here, we use in vivo lineage tracing with DiI, together with neuronal differentiation markers, to establish the first detailed fate-map for placode-derived sensory neurons in a jawless fish, the sea lamprey Petromyzon marinus, and to confirm that neural crest cells in the lamprey contribute to the cranial sensory ganglia. We also show that a pan-Pax3/7 antibody labels ophthalmic trigeminal (opV, profundal) placode-derived but not maxillomandibular trigeminal (mmV) placode-derived neurons, mirroring the expression of gnathostome Pax3 and suggesting that Pax3 (and its single Pax3/7 lamprey ortholog) is a pan-vertebrate marker for opV placode-derived neurons. Unexpectedly, however, our data reveal that mmV neuron precursors are located in two separate domains at neurula stages, with opV neuron precursors sandwiched between them. The different branches of the mmV nerve are not comparable between lampreys and gnatho-stomes, and spatial segregation of mmV neuron precursor territories may be a derived feature of lampreys. Nevertheless, maxillary and mandibular neurons are spatially segregated within gnathostome mmV ganglia, suggesting that a more detailed investigation of gnathostome mmV placode development would be worthwhile. Overall, however, our results highlight the conservation of cranial peripheral sensory nervous system development across vertebrates, yielding insight into ancestral vertebrate traits. © 2013 The Authors.

Simões-Costa M, Tan-Cabugao J, Antoshechkin I, Sauka-Spengler T, Bronner ME. 2014. Transcriptome analysis reveals novel players in the cranial neural crest gene regulatory network. Genome Res, 24 (2), pp. 281-290. | Show Abstract | Read more

The neural crest is an embryonic stem cell population that gives rise to a multitude of derivatives. In particular, the cranial neural crest (CNC) is unique in its ability to contribute to both facial skeleton and peripheral ganglia. To gain further insight into the molecular underpinnings that distinguish the CNC from other embryonic tissues, we have utilized a CNC-specific enhancer as a tool to isolate a pure, region-specific NC subpopulation for transcriptional profiling. The resulting data set reveals previously unknown transcription factors and signaling pathways that may influence the CNC's ability to migrate and/or differentiate into unique derivatives. To elaborate on the CNC gene regulatory network, we evaluated the effects of knocking down known neural plate border genes and early neural crest specifier genes on selected neural crest-enriched transcripts. The results suggest that ETS1 and SOX9 may act as pan-neural crest regulators of the migratory CNC. Taken together, our analysis provides unprecedented characterization of the migratory CNC transcriptome and identifies new links in the gene regulatory network responsible for development of this critical cell population.

Bassett AR, Azzam G, Wheatley L, Tibbit C, Rajakumar T, McGowan S, Stanger N, Ewels PA, Taylor S, Ponting CP et al. 2014. Understanding functional miRNA-target interactions in vivo by site-specific genome engineering. Nat Commun, 5 pp. 4640. | Show Abstract | Read more

MicroRNA (miRNA) target recognition is largely dictated by short 'seed' sequences, and single miRNAs therefore have the potential to regulate a large number of genes. Understanding the contribution of specific miRNA-target interactions to the regulation of biological processes in vivo remains challenging. Here we use transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technologies to interrogate the functional relevance of predicted miRNA response elements (MREs) to post-transcriptional silencing in zebrafish and Drosophila. We also demonstrate an effective strategy that uses CRISPR-mediated homology-directed repair with short oligonucleotide donors for the assessment of MRE activity in human cells. These methods facilitate analysis of the direct phenotypic consequences resulting from blocking specific miRNA-MRE interactions at any point during development.

Parker HJ, Sauka-Spengler T, Bronner M, Elgar G. 2014. A reporter assay in lamprey embryos reveals both functional conservation and elaboration of vertebrate enhancers. PLoS One, 9 (1), pp. e85492. | Show Abstract | Read more

The sea lamprey is an important model organism for investigating the evolutionary origins of vertebrates. As more vertebrate genome sequences are obtained, evolutionary developmental biologists are becoming increasingly able to identify putative gene regulatory elements across the breadth of the vertebrate taxa. The identification of these regions makes it possible to address how changes at the genomic level have led to changes in developmental gene regulatory networks and ultimately to the evolution of morphological diversity. Comparative genomics approaches using sea lamprey have already predicted a number of such regulatory elements in the lamprey genome. Functional characterisation of these sequences and other similar elements requires efficient reporter assays in lamprey. In this report, we describe the development of a transient transgenesis method for lamprey embryos. Focusing on conserved non-coding elements (CNEs), we use this method to investigate their functional conservation across the vertebrate subphylum. We find instances of both functional conservation and lineage-specific functional evolution of CNEs across vertebrates, emphasising the utility of functionally testing homologous CNEs in their host species.

Amemiya CT, Alföldi J, Lee AP, Fan S, Philippe H, Maccallum I, Braasch I, Manousaki T, Schneider I, Rohner N et al. 2013. The African coelacanth genome provides insights into tetrapod evolution. Nature, 496 (7445), pp. 311-316. | Show Abstract | Read more

The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.

Smith JJ, Kuraku S, Holt C, Sauka-Spengler T, Jiang N, Campbell MS, Yandell MD, Manousaki T, Meyer A, Bloom OE et al. 2013. Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution. Nat Genet, 45 (4), pp. 415-421e2. | Show Abstract | Read more

Lampreys are representatives of an ancient vertebrate lineage that diverged from our own ∼500 million years ago. By virtue of this deeply shared ancestry, the sea lamprey (P. marinus) genome is uniquely poised to provide insight into the ancestry of vertebrate genomes and the underlying principles of vertebrate biology. Here, we present the first lamprey whole-genome sequence and assembly. We note challenges faced owing to its high content of repetitive elements and GC bases, as well as the absence of broad-scale sequence information from closely related species. Analyses of the assembly indicate that two whole-genome duplications likely occurred before the divergence of ancestral lamprey and gnathostome lineages. Moreover, the results help define key evolutionary events within vertebrate lineages, including the origin of myelin-associated proteins and the development of appendages. The lamprey genome provides an important resource for reconstructing vertebrate origins and the evolutionary events that have shaped the genomes of extant organisms.

Modrell MS, Hockman D, Uy B, Buckley D, Sauka-Spengler T, Bronner ME, Baker CV. 2014. A fate-map for cranial sensory ganglia in the sea lamprey. Dev Biol, 385 (2), pp. 405-416. | Show Abstract | Read more

Cranial neurogenic placodes and the neural crest make essential contributions to key adult characteristics of all vertebrates, including the paired peripheral sense organs and craniofacial skeleton. Neurogenic placode development has been extensively characterized in representative jawed vertebrates (gnathostomes) but not in jawless fishes (agnathans). Here, we use in vivo lineage tracing with DiI, together with neuronal differentiation markers, to establish the first detailed fate-map for placode-derived sensory neurons in a jawless fish, the sea lamprey Petromyzon marinus, and to confirm that neural crest cells in the lamprey contribute to the cranial sensory ganglia. We also show that a pan-Pax3/7 antibody labels ophthalmic trigeminal (opV, profundal) placode-derived but not maxillomandibular trigeminal (mmV) placode-derived neurons, mirroring the expression of gnathostome Pax3 and suggesting that Pax3 (and its single Pax3/7 lamprey ortholog) is a pan-vertebrate marker for opV placode-derived neurons. Unexpectedly, however, our data reveal that mmV neuron precursors are located in two separate domains at neurula stages, with opV neuron precursors sandwiched between them. The different branches of the mmV nerve are not comparable between lampreys and gnatho-stomes, and spatial segregation of mmV neuron precursor territories may be a derived feature of lampreys. Nevertheless, maxillary and mandibular neurons are spatially segregated within gnathostome mmV ganglia, suggesting that a more detailed investigation of gnathostome mmV placode development would be worthwhile. Overall, however, our results highlight the conservation of cranial peripheral sensory nervous system development across vertebrates, yielding insight into ancestral vertebrate traits.

Cited:

226

Scopus

Smith JJ, Kuraku S, Holt C, Sauka-Spengler T, Jiang N, Campbell MS, Yandell MD, Manousaki T, Meyer A, Bloom OE et al. 2013. Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution Nature Genetics, 45 (4), pp. 415-421. | Show Abstract | Read more

Lampreys are representatives of an ancient vertebrate lineage that diverged from our own ∼500 million years ago. By virtue of this deeply shared ancestry, the sea lamprey (P. marinus) genome is uniquely poised to provide insight into the ancestry of vertebrate genomes and the underlying principles of vertebrate biology. Here, we present the first lamprey whole-genome sequence and assembly. We note challenges faced owing to its high content of repetitive elements and GC bases, as well as the absence of broad-scale sequence information from closely related species. Analyses of the assembly indicate that two whole-genome duplications likely occurred before the divergence of ancestral lamprey and gnathostome lineages. Moreover, the results help define key evolutionary events within vertebrate lineages, including the origin of myelin-associated proteins and the development of appendages. The lamprey genome provides an important resource for reconstructing vertebrate origins and the evolutionary events that have shaped the genomes of extant organisms.

Simões-Costa MS, McKeown SJ, Tan-Cabugao J, Sauka-Spengler T, Bronner ME. 2012. Dynamic and differential regulation of stem cell factor FoxD3 in the neural crest is Encrypted in the genome. PLoS Genet, 8 (12), pp. e1003142. | Show Abstract | Read more

The critical stem cell transcription factor FoxD3 is expressed by the premigratory and migrating neural crest, an embryonic stem cell population that forms diverse derivatives. Despite its important role in development and stem cell biology, little is known about what mediates FoxD3 activity in these cells. We have uncovered two FoxD3 enhancers, NC1 and NC2, that drive reporter expression in spatially and temporally distinct manners. Whereas NC1 activity recapitulates initial FoxD3 expression in the cranial neural crest, NC2 activity recapitulates initial FoxD3 expression at vagal/trunk levels while appearing only later in migrating cranial crest. Detailed mutational analysis, in vivo chromatin immunoprecipitation, and morpholino knock-downs reveal that transcription factors Pax7 and Msx1/2 cooperate with the neural crest specifier gene, Ets1, to bind to the cranial NC1 regulatory element. However, at vagal/trunk levels, they function together with the neural plate border gene, Zic1, which directly binds to the NC2 enhancer. These results reveal dynamic and differential regulation of FoxD3 in distinct neural crest subpopulations, suggesting that heterogeneity is encrypted at the regulatory level. Isolation of neural crest enhancers not only allows establishment of direct regulatory connections underlying neural crest formation, but also provides valuable tools for tissue specific manipulation and investigation of neural crest cell identity in amniotes.

Hu N, Strobl-Mazzulla P, Sauka-Spengler T, Bronner ME. 2012. DNA methyltransferase3A as a molecular switch mediating the neural tube-to-neural crest fate transition. Genes Dev, 26 (21), pp. 2380-2385. | Show Abstract | Read more

Here, we explore whether silencing via promoter DNA methylation plays a role in neural versus neural crest cell lineage decisions. We show that DNA methyltransferase3A (DNMT3A) promotes neural crest specification by directly mediating repression of neural genes like Sox2 and Sox3. DNMT3A is expressed in the neural plate border, and its knockdown causes ectopic Sox2 and Sox3 expression at the expense of neural crest markers. In vivo chromatin immunoprecipitation of neural folds demonstrates that DNMT3A specifically associates with CpG islands in the Sox2 and Sox3 promoter regions, resulting in their repression by methylation. Thus, DNMT3A functions as a molecular switch, repressing neural to favor neural crest cell fate.

Uy BR, Simoes-Costa M, Sauka-Spengler T, Bronner ME. 2012. Expression of Sox family genes in early lamprey development. Int J Dev Biol, 56 (5), pp. 377-383. | Show Abstract | Read more

Members of the Sox (Sry-related high mobility group box) family of transcription factors play a variety of roles during development of both vertebrates and invertebrates. A marked expansion in gene number occurred during the emergence of vertebrates, apparently via gene duplication events that are thought to have facilitated new functions. By screening a macroarrayed library as well as the lamprey genome, we have isolated genes of the Sox B, D, E and F subfamilies in the basal jawless vertebrate, lamprey. The expression patterns of all identified Sox genes were examined from gastrulation through early organogenesis (embryonic day 4-14), with particular emphasis on the neural crest, a vertebrate innovation. Coupled with phylogenetic analysis of these Sox genes, the results provide insight into gene duplication and di-vergence in paralog deployment occurring during early vertebrate evolution.

Cited:

57

Scopus

Prasad MS, Sauka-Spengler T, LaBonne C. 2012. Induction of the neural crest state: Control of stem cell attributes by gene regulatory, post-transcriptional and epigenetic interactions Developmental Biology, 366 (1), pp. 10-21. | Show Abstract | Read more

Neural crest cells are a population of multipotent stem cell-like progenitors that arise at the neural plate border in vertebrates, migrate extensively, and give rise to diverse derivatives such as melanocytes, craniofacial cartilage and bone, smooth muscle, peripheral and enteric neurons and glia. The neural crest gene regulatory network (NC-GRN) includes a number of key factors that are used reiteratively to control multiple steps in the development of neural crest cells, including the acquisition of stem cell attributes. It is therefore essential to understand the mechanisms that control the distinct functions of such reiteratively used factors in different cellular contexts. The context-dependent control of neural crest specification is achieved through combinatorial interaction with other factors, post-transcriptional and post-translational modifications, and the epigenetic status and chromatin state of target genes. Here we review the current understanding of the NC-GRN, including the role of the neural crest specifiers, their links to the control of "stemness," and their dynamic context-dependent regulation during the formation of neural crest progenitors. © 2012 Elsevier Inc.

Betancur P, Sauka-Spengler T, Bronner M. 2011. A Sox10 enhancer element common to the otic placode and neural crest is activated by tissue-specific paralogs. Development, 138 (17), pp. 3689-3698. | Show Abstract | Read more

The otic placode, a specialized region of ectoderm, gives rise to components of the inner ear and shares many characteristics with the neural crest, including expression of the key transcription factor Sox10. Here, we show that in avian embryos, a highly conserved cranial neural crest enhancer, Sox10E2, also controls the onset of Sox10 expression in the otic placode. Interestingly, we show that different combinations of paralogous transcription factors (Sox8, Pea3 and cMyb versus Sox9, Ets1 and cMyb) are required to mediate Sox10E2 activity in the ear and neural crest, respectively. Mutating their binding motifs within Sox10E2 greatly reduces enhancer activity in the ear. Moreover, simultaneous knockdown of Sox8, Pea3 and cMyb eliminates not only the enhancer-driven reporter expression, but also the onset of endogenous Sox10 expression in the ear. Rescue experiments confirm that the specific combination of Myb together with Sox8 and Pea3 is responsible for the onset of Sox10 expression in the otic placode, as opposed to Myb plus Sox9 and Ets1 for neural crest Sox10 expression. Whereas SUMOylation of Sox8 is not required for the initial onset of Sox10 expression, it is necessary for later otic vesicle formation. This new role of Sox8, Pea3 and cMyb in controlling Sox10 expression via a common otic/neural crest enhancer suggests an evolutionarily conserved function for the combination of paralogous transcription factors in these tissues of distinct embryological origin.

Parker HJ, Piccinelli P, Sauka-Spengler T, Bronner M, Elgar G. 2011. Ancient Pbx-Hox signatures define hundreds of vertebrate developmental enhancers. BMC Genomics, 12 (1), pp. 637. | Show Abstract | Read more

BACKGROUND: Gene regulation through cis-regulatory elements plays a crucial role in development and disease. A major aim of the post-genomic era is to be able to read the function of cis-regulatory elements through scrutiny of their DNA sequence. Whilst comparative genomics approaches have identified thousands of putative regulatory elements, our knowledge of their mechanism of action is poor and very little progress has been made in systematically de-coding them. RESULTS: Here, we identify ancient functional signatures within vertebrate conserved non-coding elements (CNEs) through a combination of phylogenetic footprinting and functional assay, using genomic sequence from the sea lamprey as a reference. We uncover a striking enrichment within vertebrate CNEs for conserved binding-site motifs of the Pbx-Hox hetero-dimer. We further show that these predict reporter gene expression in a segment specific manner in the hindbrain and pharyngeal arches during zebrafish development. CONCLUSIONS: These findings evoke an evolutionary scenario in which many CNEs evolved early in the vertebrate lineage to co-ordinate Hox-dependent gene-regulatory interactions that pattern the vertebrate head. In a broader context, our evolutionary analyses reveal that CNEs are composed of tightly linked transcription-factor binding-sites (TFBSs), which can be systematically identified through phylogenetic footprinting approaches. By placing a large number of ancient vertebrate CNEs into a developmental context, our findings promise to have a significant impact on efforts toward de-coding gene-regulatory elements that underlie vertebrate development, and will facilitate building general models of regulatory element evolution.

Häming D, Simoes-Costa M, Uy B, Valencia J, Sauka-Spengler T, Bronner-Fraser M. 2011. Expression of sympathetic nervous system genes in lamprey suggests their recruitment for specification of a new vertebrate feature PLoS ONE, 6 (10),

Häming D, Simoes-Costa M, Uy B, Valencia J, Sauka-Spengler T, Bronner-Fraser M. 2011. Expression of sympathetic nervous system genes in Lamprey suggests their recruitment for specification of a new vertebrate feature. PLoS One, 6 (10), pp. e26543. | Show Abstract | Read more

The sea lamprey is a basal, jawless vertebrate that possesses many neural crest derivatives, but lacks jaws and sympathetic ganglia. This raises the possibility that the factors involved in sympathetic neuron differentiation were either a gnathostome innovation or already present in lamprey, but serving different purposes. To distinguish between these possibilities, we isolated lamprey homologues of transcription factors associated with peripheral ganglion formation and examined their deployment in lamprey embryos. We further performed DiI labeling of the neural tube combined with neuronal markers to test if neural crest-derived cells migrate to and differentiate in sites colonized by sympathetic ganglia in jawed vertebrates. Consistent with previous anatomical data in adults, our results in lamprey embryos reveal that neural crest cells fail to migrate ventrally to form sympathetic ganglia, though they do form dorsal root ganglia adjacent to the neural tube. Interestingly, however, paralogs of the battery of transcription factors that mediate sympathetic neuron differentiation (dHand, Ascl1 and Phox2b) are present in the lamprey genome and expressed in various sites in the embryo, but fail to overlap in any ganglionic structures. This raises the intriguing possibility that they may have been recruited during gnathostome evolution to a new function in a neural crest derivative.

Betancur P, Bronner-Fraser M, Sauka-Spengler T. 2010. Assembling neural crest regulatory circuits into a gene regulatory network. Annu Rev Cell Dev Biol, 26 (1), pp. 581-603. | Show Abstract | Read more

The neural crest is a multipotent stem cell&#x2013;like population that gives rise to a wide range of derivatives in the vertebrate embryo including elements of the craniofacial skeleton and peripheral nervous system as well as melanocytes. The neural crest forms in a series of regulatory steps that include induction and specification of the prospective neural crest territory&#x2013;neural plate border, specification of bona fide neural crest progenitors, and differentiation into diverse derivatives. These individual processes during neural crest ontogeny are controlled by regulatory circuits that can be assembled into a hierarchical gene regulatory network (GRN). Here we present an overview of the GRN that orchestrates the formation of cranial neural crest cells. Formulation of this network relies on information largely inferred from gene perturbation studies performed in several vertebrate model organisms. Our representation of the cranial neural crest GRN also includes information about direct regulatory interactions obtained from the cis-regulatory analyses performed to date, which increases the resolution of the architectural circuitry within the network.

Sauka-Spengler T, Bronner M. 2010. Snapshot: neural crest. Cell, 143 (3), pp. 486-486.e1. | Read more

Cerny R, Cattell M, Sauka-Spengler T, Bronner-Fraser M, Yu F, Medeiros DM. 2010. Evidence for the prepattern/cooption model of vertebrate jaw evolution. Proc Natl Acad Sci U S A, 107 (40), pp. 17262-17267. | Show Abstract | Read more

The appearance of jaws was a turning point in vertebrate evolution because it allowed primitive vertebrates to capture and process large, motile prey. The vertebrate jaw consists of separate dorsal and ventral skeletal elements connected by a joint. How this structure evolved from the unjointed gill bar of a jawless ancestor is an unresolved question in vertebrate evolution. To understand the developmental bases of this evolutionary transition, we examined the expression of 12 genes involved in vertebrate pharyngeal patterning in the modern jawless fish lamprey. We find nested expression of Dlx genes, as well as combinatorial expression of Msx, Hand and Gsc genes along the dorso-ventral (DV) axis of the lamprey pharynx, indicating gnathostome-type pharyngeal patterning evolved before the appearance of the jaw. In addition, we find that Bapx and Gdf5/6/7, key regulators of joint formation in gnathostomes, are not expressed in the lamprey first arch, whereas Barx, which is absent from the intermediate first arch in gnathostomes, marks this domain in lamprey. Taken together, these data support a new scenario for jaw evolution in which incorporation of Bapx and Gdf5/6/7 into a preexisting DV patterning program drove the evolution of the jaw by altering the identity of intermediate first-arch chondrocytes. We present this "Pre-pattern/Cooption" model as an alternative to current models linking the evolution of the jaw to the de novo appearance of sophisticated pharyngeal DV patterning.

Strobl-Mazzulla PH, Sauka-Spengler T, Bronner-Fraser M. 2010. Histone demethylase JmjD2A regulates neural crest specification. Dev Cell, 19 (3), pp. 460-468. | Show Abstract | Read more

The neural crest is a multipotent stem cell-like population that is induced during gastrulation, but only acquires its characteristic morphology, migratory ability, and gene expression profile after neurulation. This raises the intriguing possibility that precursors are actively maintained by epigenetic influences in a stem cell-like state. Accordingly, we report that dynamic histone modifications are critical for proper temporal control of neural crest gene expression in vivo. The histone demethylase, JumonjiD2A (JmjD2A/KDM4A), is expressed in the forming neural folds. Loss of JmjD2A function causes dramatic downregulation of neural crest specifier genes analyzed by multiplex NanoString and in situ hybridization. Importantly, in vivo chromatin immunoprecipitation reveals direct stage-specific interactions of JmjD2A with regulatory regions of neural crest genes, and associated temporal modifications in methylation states of lysine residues directly affected by JmjD2A activity. Our findings show that chromatin modifications directly control neural crest genes in vertebrate embryos via modulating histone methylation.

Smith JJ, Stuart AB, Sauka-Spengler T, Clifton SW, Amemiya CT. 2010. Development and analysis of a germline BAC resource for the sea lamprey, a vertebrate that undergoes substantial chromatin diminution. Chromosoma, 119 (4), pp. 381-389. | Show Abstract | Read more

Over the last several years, the sea lamprey (Petromyzon marinus) has grown substantially as a model for understanding the evolutionary fundaments and capacity of vertebrate developmental and genome biology. Recent work on the lamprey genome has resulted in a preliminary assembly of the lamprey genome and led to the realization that nearly all somatic cell lineages undergo extensive programmed rearrangements. Here we describe the development of a bacterial artificial chromosome (BAC) resource for lamprey germline DNA and use sequence information from this resource to probe the subchromosomal structure of the lamprey genome. The arrayed germline BAC library represents approximately 10x coverage of the lamprey genome. Analyses of BAC-end sequences reveal that the lamprey genome possesses a high content of repetitive sequences (relative to human), which show strong clustering at the subchromosomal level. This pattern is not unexpected given that the sea lamprey genome is dispersed across a large number of chromosomes (n approximately 99) and suggests a low-copy DNA targeting strategy for efficiently generating informative paired-BAC-end linkages from highly repetitive genomes. This library therefore represents a new and biologically informed resource for understanding the structure of the lamprey genome and the biology of programmed genome rearrangement.

Betancur P, Bronner-Fraser M, Sauka-Spengler T. 2010. Genomic code for Sox10 activation reveals a key regulatory enhancer for cranial neural crest. Proc Natl Acad Sci U S A, 107 (8), pp. 3570-3575. | Show Abstract | Read more

The neural crest is a multipotent, stem cell-like population that migrates extensively in the embryo and forms a wide array of derivatives, ranging from neurons to melanocytes and cartilage. Analyses of the gene regulatory network driving neural crest development revealed Sox10 as one of the earliest neural crest-specifying genes, cell-autonomously driving delamination and directly regulating numerous downstream effectors and differentiation gene batteries. In search of direct inputs to the neural crest specifier module, we dissected the chick Sox10 genomic region and isolated two downstream regulatory regions with distinct spatiotemporal activity. A unique element, Sox10E2 represents the earliest-acting neural crest cis-regulatory element, critical for initiating Sox10 expression in newly formed cranial, but not vagal and trunk neural crest. A second element, Sox10E1, acts in later migrating vagal and trunk crest cells. Deep characterization of Sox10E2 reveals Sox9, Ets1, and cMyb as direct inputs mediating enhancer activity. ChIP, DNA-pull down, and gel-shift assays demonstrate their direct binding to the Sox10E2 enhancer in vivo, whereas mutation of their corresponding binding sites, or inactivation of the three upstream regulators, abolishes both reporter and endogenous Sox10 expression. Using cis-regulatory analysis as a tool, our study makes critical connections within the neural crest gene regulatory network, thus being unique in establishing a direct link of upstream effectors to a key neural crest specifier.

Nikitina N, Sauka-Spengler T, Bronner-Fraser M. 2009. Chapter 1. Gene regulatory networks in neural crest development and evolution. Curr Top Dev Biol, 86 pp. 1-14. | Show Abstract | Read more

The neural crest is a multipotent migratory embryonic cell population that is present in all vertebrates, but missing from basal chordates. In this chapter, we discuss recent work in amphioxus, ascidians, lamprey, and gnathostomes that reflects the current state of knowledge of the evolutionary origin of this fascinating cell population. We summarize recent evidence for the ongoing diversification of the neural crest in several vertebrate species, with particular reference to studies in nontraditional vertebrate model organisms.

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Nikitina N, Bronner-Fraser M, Sauka-Spengler T. 2009. The sea lamprey Petromyzon marinus: A model for evolutionary and developmental biology Cold Spring Harbor Protocols, 4 (1), | Read more

Nikitina N, Bronner-Fraser M, Sauka-Spengler T. 2009. The sea lamprey Petromyzon marinus: a model for evolutionary and developmental biology. Cold Spring Harb Protoc, 2009 (1), pp. pdb.emo113. | Show Abstract | Read more

Sea lampreys (Petromyzon marinus) are cyclostomes, the most basal extant group of vertebrates, and are thought to have existed largely unchanged for more than 500 million years. They are aquatic, eel-shaped animals that spend a major part of their life as filter-feeding larvae called ammocoetes, inhabiting many freshwater bodies in the northern hemisphere. After metamorphosis, sea lampreys migrate to the ocean (or to the Great Lakes), where they feed on the blood and bodily fluids of salmonid fish and ultimately return to freshwater streams and rivers to spawn and die. The unique evolutionary position of lampreys and the relative ease of obtaining mature adults and embryos make this animal an ideal model for investigations into early vertebrate evolution. Studies of features shared between lampreys and jawed vertebrates, but distinct from those in nonvertebrate chordates, have provided information on the origin and evolution of hallmark vertebrate characteristics such as the neural crest, ectodermal placodes, and jaw. In addition, studies of features that are unique to lampreys (e.g., the variable lymphocyte receptor-mediated immune system) provide insights into mechanisms of parallel evolution (e.g., the adaptive immune system). With the establishment of techniques for the extended maintenance and spawning of lampreys in the laboratory, the sequencing of the lamprey genome, and the adaptation and optimization of many established molecular biology and histochemistry techniques for use in this species, P. marinus is poised to become an evolutionary developmental model of choice.

Nikitina N, Sauka-Spengler T, Bronner-Fraser M. 2008. Dissecting early regulatory relationships in the lamprey neural crest gene network. Proc Natl Acad Sci U S A, 105 (51), pp. 20083-20088. | Show Abstract | Read more

The neural crest, a multipotent embryonic cell type, originates at the border between neural and nonneural ectoderm. After neural tube closure, these cells undergo an epithelial-mesenchymal transition, migrate to precise, often distant locations, and differentiate into diverse derivatives. Analyses of expression and function of signaling and transcription factors in higher vertebrates has led to the proposal that a neural crest gene regulatory network (NC-GRN) orchestrates neural crest formation. Here, we interrogate the NC-GRN in the lamprey, taking advantage of its slow development and basal phylogenetic position to resolve early inductive events, 1 regulatory step at the time. To establish regulatory relationships at the neural plate border, we assess relative expression of 6 neural crest network genes and effects of individually perturbing each on the remaining 5. The results refine an upstream portion of the NC-GRN and reveal unexpected order and linkages therein; e.g., lamprey AP-2 appears to function early as a neural plate border rather than a neural crest specifier and in a pathway linked to MsxA but independent of ZicA. These findings provide an ancestral framework for performing comparative tests in higher vertebrates in which network linkages may be more difficult to resolve because of their rapid development.

Sauka-Spengler T, Bronner-Fraser M. 2008. Evolution of the neural crest viewed from a gene regulatory perspective. Genesis, 46 (11), pp. 673-682. | Show Abstract | Read more

Neural crest cells are a vertebrate innovation and form a wide variety of embryonic cell types as diverse as peripheral neurons and facial skeleton. They undergo complex migration and differentiation processes from their site of origin in the developing central nervous system to their final destinations in the periphery. In this review, we summarize recent data on the current formulation of a gene regulatory network underlying neural crest formation and its roots at the base of the vertebrate lineage. Analyzing neural crest formation from a gene regulatory viewpoint provides insights into both the developmental mechanisms and evolutionary origins of this vertebrate-specific cell type.

Sauka-Spengler T, Bronner-Fraser M. 2008. A gene regulatory network orchestrates neural crest formation. Nat Rev Mol Cell Biol, 9 (7), pp. 557-568. | Show Abstract | Read more

The neural crest is a multipotent, migratory cell population that is unique to vertebrate embryos and gives rise to many derivatives, ranging from the peripheral nervous system to the craniofacial skeleton and pigment cells. A multimodule gene regulatory network mediates the complex process of neural crest formation, which involves the early induction and maintenance of the precursor pool, emigration of the neural crest progenitors from the neural tube via an epithelial to mesenchymal transition, migration of progenitor cells along distinct pathways and overt differentiation into diverse cell types. Here, we review our current understanding of these processes and discuss the molecular players that are involved in the neural crest gene regulatory network.

Sauka-Spengler T, Bronner-Fraser M. 2008. Insights from a sea lamprey into the evolution of neural crest gene regulatory network. Biol Bull, 214 (3), pp. 303-314. | Show Abstract | Read more

The neural crest is a vertebrate innovation that forms at the embryonic neural plate border, transforms from epithelial to mesenchymal, migrates extensively throughout the embryo along well-defined pathways, and differentiates into a plethora of derivatives that include elements of peripheral nervous system, craniofacial skeleton, melanocytes, etc. The complex process of neural crest formation is guided by multiple regulatory modules that define neural crest gene regulatory network (NC GRN), which allows the neural crest to progressively acquire all of its defining characteristics. The molecular study of neural crest formation in lamprey, a basal extant vertebrate, consisting in identification and functional tests of molecular elements at each regulatory level of this network, has helped address the question of the timing of emergence of NC GRN and define its basal state. The results have revealed striking conservation in deployment of upstream factors and regulatory modules, suggesting that proximal portions of the network arose early in vertebrate evolution and have been tightly conserved for more than 500 million years. In contrast, certain differences were observed in deployment of some neural crest specifier and downstream effector genes expected to confer species-specific migratory and differentiation properties.

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Sauka-Spengler T, Barembaum M. 2008. Chapter 12 Gain- and Loss-of-Function Approaches in the Chick Embryo Methods in Cell Biology, 87 pp. 237-256. | Show Abstract | Read more

The chicken embryo has been used as a classical embryological model for studying developmental events because of its ready availability, similarity to the human embryos, and amenability to embryological and surgical manipulations. With the arrival of the molecular era, however, avian embryos presented distinct experimental limitations, largely because of the difficulty of performing targeted mutagenesis or transgenic studies. However, in the last decade and a half, a number of new methods for transient transgenesis have been developed that allow efficient alteration of gene function during early embryonic development. These techniques have made it possible to study the effects of gene inactivation or overexpression on downstream transcriptional regulation as well as on embryonic derivatives. This, together with sequencing of the chicken genome, has allowed the chicken embryo to enter the genomic era. While attempts to establish germ line transgenesis are ongoing, methods for rapid, transient spatiotemporally targeted gene alterations have thus again re-established the chick embryo as an important experimental niche by making it possible to apply genetics in concert with classical embryological techniques. This provides a unique tool to explore the role of developmentally important genes (Ishii and Mikawa, 2005; Itasaki et al., 1999; Krull, 2004; Ogura, 2002; Swartz et al., 2001). Transient transfection methods have allowed for efficient mis- and overexpression of transgenes. For long-term analyses, retrovirally mediated gene transfer has particular advantage. For short-term experiments, electroporation and adenoviral-mediated gene transfer methods provide transient expression, largely because of the short persistence time of the transgene within the cell. More recently, Tol2 transposon-mediated constructs have been employed, allowing for integration into the genome and prolonged expression of the transgene (Sato et al., 2007), see Chapter 14 by Takahashi et al., this volume). These methods today are routinely used for gain-of-function analysis, to overexpress or ectopically express genes of interest (Arber et al., 1999; Barembaum and Bronner-Fraser, 2007; Bel-Vialar et al., 2002). Loss-of-function experiments are also possible using electroporation of dominant-negative constructs that act as competitive inhibitors (Bel-Vialar et al., 2002; Renzi et al., 2000; Suzuki-Hirano et al., 2005), morpholino antisense oligos (Basch et al., 2006; Kos et al., 2001; Sheng et al., 2003) that block translation or splicing, or constructs expressing small interfering or small hairpin RNAs (siRNAs or shRNAs) (Chesnutt and Niswander, 2004; Das et al., 2006; Katahira and Nakamura, 2003). Electroporation as the most popular method of the transient transfection into the chick embryos. Electroporation of chicken embryos involves application of an electric field to the exposed tissue that transiently disrupts the stability of the cell plasma membrane, creating reversible pores through which nucleic acids or their analogues can be readily transported into the cytosol. The use of this method for transfection into the vertebrate embryos has been facilitated by adapting the voltage parameters and the type and the duration of the electric pulse. By applying several successive square pulses at a very low voltage, with long rest periods in between, one can successfully deliver a DNA construct or another small charged particle into the cytoplasm, with minimal cell death, high efficiency of the uptake and good embryonic survival rate. The size limit of the DNA molecule that can be transfected in such a way is not yet known, though it is more likely that the size limitation in this procedure (if any) lies within the practical problems of cloning large fragments into the plasmid. We routinely overexpress constructs containing 3-4 kb inserts and coharboring a GFP or RFP reporter whose translation is initiated from an internal ribosomal entry site (IRES), thus allowing easy detection of the electroporated cells. © 2008 Elsevier Inc. All rights reserved.

Coolen M, Nicolle D, Plouhinec JL, Gombault A, Sauka-Spengler T, Menuet A, Pieau C, Mazan S. 2008. Molecular characterization of the gastrula in the turtle Emys orbicularis: an evolutionary perspective on gastrulation. PLoS One, 3 (7), pp. e2676. | Show Abstract | Read more

Due to the presence of a blastopore as in amphibians, the turtle has been suggested to exemplify a transition form from an amphibian- to an avian-type gastrulation pattern. In order to test this hypothesis and gain insight into the emergence of the unique characteristics of amniotes during gastrulation, we have performed the first molecular characterization of the gastrula in a reptile, the turtle Emys orbicularis. The study of Brachyury, Lim1, Otx2 and Otx5 expression patterns points to a highly conserved dynamic of expression with amniote model organisms and makes it possible to identify the site of mesoderm internalization, which is a long-standing issue in reptiles. Analysis of Brachyury expression also highlights the presence of two distinct phases, less easily recognizable in model organisms and respectively characterized by an early ring-shaped and a later bilateral symmetrical territory. Systematic comparisons with tetrapod model organisms lead to new insights into the relationships of the blastopore/blastoporal plate system shared by all reptiles, with the blastopore of amphibians and the primitive streak of birds and mammals. The biphasic Brachyury expression pattern is also consistent with recent models of emergence of bilateral symmetry, which raises the question of its evolutionary significance.

Putnam NH, Butts T, Ferrier DE, Furlong RF, Hellsten U, Kawashima T, Robinson-Rechavi M, Shoguchi E, Terry A, Yu JK et al. 2008. The amphioxus genome and the evolution of the chordate karyotype. Nature, 453 (7198), pp. 1064-1071. | Show Abstract | Read more

Lancelets ('amphioxus') are the modern survivors of an ancient chordate lineage, with a fossil record dating back to the Cambrian period. Here we describe the structure and gene content of the highly polymorphic approximately 520-megabase genome of the Florida lancelet Branchiostoma floridae, and analyse it in the context of chordate evolution. Whole-genome comparisons illuminate the murky relationships among the three chordate groups (tunicates, lancelets and vertebrates), and allow not only reconstruction of the gene complement of the last common chordate ancestor but also partial reconstruction of its genomic organization, as well as a description of two genome-wide duplications and subsequent reorganizations in the vertebrate lineage. These genome-scale events shaped the vertebrate genome and provided additional genetic variation for exploitation during vertebrate evolution.

Sauka-Spengler T, Meulemans D, Jones M, Bronner-Fraser M. 2007. Ancient evolutionary origin of the neural crest gene regulatory network. Dev Cell, 13 (3), pp. 405-420. | Show Abstract | Read more

The vertebrate neural crest migrates from its origin, the neural plate border, to form diverse derivatives. We previously hypothesized that a neural crest gene regulatory network (NC-GRN) guides neural crest formation. Here, we investigate when during evolution this hypothetical network emerged by analyzing neural crest formation in lamprey, a basal extant vertebrate. We identify 50 NC-GRN homologs and use morpholinos to demonstrate a critical role for eight transcriptional regulators. The results reveal conservation in deployment of upstream factors, suggesting that proximal portions of the network arose early in vertebrate evolution and have been conserved for >500 million years. We found biphasic expression of neural crest specifiers and differences in deployment of some specifiers and effectors expected to confer species-specific properties. By testing the collective expression and function of neural crest genes in a single, basal vertebrate, we reveal the ground state of the NC-GRN and resolve ambiguities between model organisms.

Coolen M, Sauka-Spengler T, Nicolle D, Le-Mentec C, Lallemand Y, Da Silva C, Plouhinec JL, Robert B, Wincker P, Shi DL, Mazan S. 2007. Evolution of axis specification mechanisms in jawed vertebrates: insights from a chondrichthyan. PLoS One, 2 (4), pp. e374. | Show Abstract | Read more

The genetic mechanisms that control the establishment of early polarities and their link with embryonic axis specification and patterning seem to substantially diverge across vertebrates. In amphibians and teleosts, the establishment of an early dorso-ventral polarity determines both the site of axis formation and its rostro-caudal orientation. In contrast, amniotes retain a considerable plasticity for their site of axis formation until blastula stages and rely on signals secreted by extraembryonic tissues, which have no clear equivalents in the former, for the establishment of their rostro-caudal pattern. The rationale for these differences remains unknown. Through detailed expression analyses of key development genes in a chondrichthyan, the dogfish Scyliorhinus canicula, we have reconstructed the ancestral pattern of axis specification in jawed vertebrates. We show that the dogfish displays compelling similarities with amniotes at blastula and early gastrula stages, including the presence of clear homologs of the hypoblast and extraembryonic ectoderm. In the ancestral state, these territories are specified at opposite poles of an early axis of bilateral symmetry, homologous to the dorso-ventral axis of amphibians or teleosts, and aligned with the later forming embryonic axis, from head to tail. Comparisons with amniotes suggest that a dorsal expansion of extraembryonic ectoderm, resulting in an apparently radial symmetry at late blastula stages, has taken place in their lineage. The synthesis of these results with those of functional analyses in model organisms supports an evolutionary link between the dorso-ventral polarity of amphibians and teleosts and the embryonic-extraembryonic organisation of amniotes. It leads to a general model of axis specification in gnathostomes, which provides a comparative framework for a reassessment of conservations both among vertebrates and with more distant metazoans.

Sauka-Spengler T, Bronner-Fraser M. 2006. Development and evolution of the migratory neural crest: a gene regulatory perspective. Curr Opin Genet Dev, 16 (4), pp. 360-366. | Show Abstract | Read more

The neural crest, a uniquely vertebrate characteristic, gives rise to pigment cells, much of the peripheral nervous system, the craniofacial skeleton, and a plethora of other cell types. Classical embryological studies have revealed important details about the migratory pathways followed by these cells, and their subsequent differentiation into diverse derivatives. More recently, many aspects of the molecular cascade of events involved in neural crest induction and generation of these migratory cells have been revealed. Formation of the neural crest appears to involve a network of interactions whereby signaling molecules initiate the induction and, subsequently, the establishment of the neural plate border, which is marked by expression of a characteristic set of transcription factors designated as neural plate border-specifiers. These in turn regulate other transcription factors termed neural crest-specifiers, which control genes involved in neural crest delamination, the generation of migratory cells and ultimately the acquisition of appropriate fates.

Plouhinec JL, Leconte L, Sauka-Spengler T, Bovolenta P, Mazan S, Saule S. 2005. Comparative analysis of gnathostome Otx gene expression patterns in the developing eye: implications for the functional evolution of the multigene family. Dev Biol, 278 (2), pp. 560-575. | Show Abstract | Read more

We have performed a detailed analysis of the expression pattern of the three gnathostome Otx classes in order to gain new insights into their functional evolution. Expression patterns were examined in the developing eye of a chondrichthyan, the dogfish, and an amniote, the chick, and compared with the capacity of paralogous proteins to induce a pigmented phenotype in cultured retina cells in cooperation with the bHLH-leucine zipper protein Mitf. This analysis indicates that each Otx class is characterized by highly specific and conserved expression features in the presumptive RPE, where Otx1 and Otx2, but not Otx5, are transcribed at optic vesicle stages, in the differentiating neural retina, where Otx2 and Otx5 show a conserved dynamic expression pattern, and in the forming ciliary process, a major site of Otx1 expression. Furthermore, the paralogous proteins of the dogfish and the mouse do not display any significant difference in their capacity to induce a pigmented phenotype, suggesting a functional equivalency in the specification and differentiation of the RPE. These data indicate that specific functions selectively involving each Otx orthology class were fixed prior to the gnathostome radiation and highlight the prominent role of regulatory changes in the functional diversification of the multigene family.

Fraser PE, Sauka-Spengler T. 2004. Expression of the polycomb group gene bmi-1 in the early chick embryo. Gene Expr Patterns, 5 (1), pp. 23-27. | Show Abstract | Read more

Bmi-1 is a protooncogene of the polycomb group that has important functions in self-renewal of adult stem cells and maintenance of anterior identity along the embryonic body axis. Despite these important functions, surprisingly little is known about its distribution pattern in the embryo. Here, we have isolated the chick homologue of bmi-1 and examined its expression pattern from gastrulation through neurulation and establishment of organ rudiments. During gastrulation, bmi-1 transcripts were expressed in the epiblast cells adjacent to the primitive streak. During early nervous system development, robust expression was observed in the open neural plate and later in the dorsal neural tube and much of the brain. Bmi-1 expression was also present in the developing heart primordia and the sensory placodes. The data show that bmi-1 is present in, but not restricted to, tissue containing multipotent precursor cells.

Sauka-Spengler T, Baratte B, Lepage M, Mazan S. 2003. Characterization of Brachyury genes in the dogfish S. canicula and the lamprey L. fluviatilis. Insights into gastrulation in a chondrichthyan. Dev Biol, 263 (2), pp. 296-307. | Show Abstract | Read more

In order to gain insights into the evolution of gastrulation mechanisms among vertebrates, we have characterized a Brachyury-related gene in a lamprey, Lampetra fluviatilis, and in a chondrichthyan, Scyliorhinus canicula. These two genes, respectively termed LfT and ScT, share with their osteichthyan counterparts prominent expression sites in the developing notochord, the tailbud, but also a transient expression in the prechordal plate, which is likely to be ancestral among vertebrates. In addition, the lamprey LfT gene is transcribed in the endoderm of the pharyngeal arches and the epiphysis, two expression sites that have not been reported thus far in gnathostomes, and, as in the chick, in the differentiating nephrotomes. Since Brachyury expression in nascent mesoderm and endoderm is highly conserved among vertebrates as well as cephalochordates, we have used this marker to identify these cell populations during gastrulation in the dogfish. The results suggest that these cells are initially present over the whole margin of the blastoderm and are displaced during gastrulation to its posterior part, which may correspond to the site of mesoderm and endoderm internalization. These data provide the first molecular characterization of gastrulation in a chondrichthyan. They indicate that gastrulation in the dogfish and in some amniotes shares striking similarities despite the phylogenetic distance between these species. This supports the hypothesis that the extensively divergent morphologies of gastrulae among vertebrates largely result from adaptations to the presence of yolk.

Plouhinec JL, Sauka-Spengler T, Germot A, Le Mentec C, Cabana T, Harrison G, Pieau C, Sire JY, Véron G, Mazan S. 2003. The mammalian Crx genes are highly divergent representatives of the Otx5 gene family, a gnathostome orthology class of orthodenticle-related homeogenes involved in the differentiation of retinal photoreceptors and circadian entrainment. Mol Biol Evol, 20 (4), pp. 513-521. | Show Abstract | Read more

The mammalian Crx genes are highly divergent orthodenticle (otd)-related homeogenes that play important roles in the differentiation of retinal photoreceptors and the circadian entrainment. However, their evolutionary origin and orthological relationships with other otd-related genes remain unclear. An orthology relationship of these genes with the highly conserved Otx5 genes identified in fish and amphibians, and also expressed in the eye and epiphysis, has been proposed previously but remains controversial. To test this hypothesis, we have identified Crx genes in a wide range of mammals, including three marsupials, and Otx5-related genes in a lizard, a turtle, and two archosaurs (crocodile and chick), as well as in the pufferfish. Phylogenetic analyses of the coding sequences show that the mammalian Crx genes are orthologous to the Otx5-related genes isolated in other gnathostomes. They also indicate that a duplication event has taken place in actinopterygians, after the splitting of the Cladistia, and that a relaxation of the structural constraints acting on the gene coding region has occurred early in the mammalian lineage. This process may be linked not only to the loss of ancestral Otx5/Crx functions during gastrulation or in the retinal pigmented epithelium, but also to the evolution of photic entrainment mechanisms in mammals.

Sauka-Spengler T, Le Mentec C, Lepage M, Mazan S. 2002. Embryonic expression of Tbx1, a DiGeorge syndrome candidate gene, in the lamprey Lampetra fluviatilis. Gene Expr Patterns, 2 (1-2), pp. 99-103. | Show Abstract | Read more

We report the embryonic expression in the lamprey Lampetra fluviatilis of Tbx1, the main candidate gene involved in DiGeorge/velo-cardio-facial syndrome (DGS/VCFS). From the end of neurulation to stage 26, Tbx1 becomes progressively expressed in all developing pharyngeal arches, as they form. Transcripts are mainly restricted to the mesodermal core and to the posterior pharyngeal endoderm, excluding ingressing neural crest cells. They are also present in the otic vesicle, in a ventral and posterior location. From a later stage (stage 27) onwards, additional expression domains in the head mesenchyme, later contributing to labial muscle precursors, and in the cloacal region, become visible. The comparison of these data with those reported in the chick and the mouse indicates a high conservation of Tbx1 expression in the pharyngeal arches among vertebrates.

Sauka-Spengler T, Germot A, Shi DL, Mazan S. 2002. Expression patterns of an Otx2 and an Otx5 orthologue in the urodele Pleurodeles waltl: implications on the evolutionary relationships between the balancers and cement gland in amphibians. Dev Genes Evol, 212 (8), pp. 380-387. | Show Abstract | Read more

We report the characterization of an Otx2 and an Otx5 orthologue in the urodele Pleurodeles waltl. These two genes, termed PwOtx2 and PwOtx5, share highly conserved expression domains with their gnathostome counterparts at tailbud stages, like the developing forebrain ( PwOtx2), or the embryonic eye and epiphysis ( PwOtx5). As in Xenopus laevis, both are also transcribed in the dorsal lip of the blastopore during gastrulation and in anterior parts of the neural plate during neurulation. In addition, PwOtx5 displays a prominent expression in the developing balancers and the lateral non-neural ectoderm during neurulation, from which they derive. By contrast, PwOtx2 expression remains undetectable in the balancers and their presumptive territory. These data suggest that PwOtx5, but not PwOtx2, may be involved in the differentiation and early specification of balancers. Comparisons of Otx5 expression patterns in P. waltland X. laevis embryos suggest that, as previously shown for Otx2, changes in the regulatory mechanisms controlling Otx5 early expression in the non-neural ectoderm may occur frequently among amphibians. These changes may be related to the rise of cement glands in anurans and of balancers in urodeles. This hypothesis could account for some similarities between the two organs, but does not support a homology relationship between them.

Derobert Y, Plouhinec JL, Sauka-Spengler T, Le Mentec C, Baratte B, Jaillard D, Mazan S. 2002. Structure and expression of three Emx genes in the dogfish Scyliorhinus canicula: functional and evolutionary implications. Dev Biol, 247 (2), pp. 390-404. | Show Abstract | Read more

We report the characterization of three Emx genes in a chondrichthyan, the dogfish Scyliorhinus canicula. Comparisons of these genes with their osteichthyan counterparts indicate that the gnathostome Emx genes belong to three distinct orthology classes, each containing one of the dogfish genes and either the tetrapod Emx1 genes (Emx1 class), the osteichthyan Emx2 genes (Emx2 class) or the zebrafish Emx1 gene (Emx3 class). While the three classes could be retrieved from the pufferfish genome data, no indication of an Emx3-related gene in tetrapods could be found in the databases, suggesting that this class may have been lost in this taxon. Expression pattern comparisons of the three dogfish Emx genes and their osteichthyan counterparts indicate that not only telencephalic, but also diencephalic Emx expression territories are highly conserved among gnathostomes. In particular, all gnathostomes share an early, dynamic phase of Emx expression, spanning presumptive dorsal diencephalic territories, which involves Emx3 in the dogfish, but another orthology class, Emx2, in tetrapods. In addition, the dogfish Emx2 gene shows a highly specific expression domain in the cephalic paraxial mesoderm from the end of gastrulation and throughout neurulation, which suggests a role in the segmentation of the cephalic mesoderm.

Sauka-Spengler T, Baratte B, Shi L, Mazan S. 2001. Structure and expression of an Otx5-related gene in the dogfish Scyliorhinus canicula: evidence for a conserved role of Otx5 and Crxgenes in the specification of photoreceptors. Dev Genes Evol, 211 (11), pp. 533-544. | Show Abstract | Read more

We report the full-length coding sequence and the expression pattern during neurulation and early organogenesis of ScOtx5, a novel member of the Otx gene family in the dogfish Scyliorhinus canicula. Phylogenetic analyses confirm that ScOtx5 is closely related to the Xenopus XlOtx5/ 5bgenes, and also to the Crx genes characterized in mammals and zebrafish. This supports the hypothesis that these genes define a third gnathostome Otx orthology class. During neurulation, ScOtx5 transcripts are detected in the foregut diverticulum and the anterior neuroectoderm. At the onset of organogenesis, ScOtx5 is transcribed over a broad domain spanning the whole prosencephalon and mesencephalon, albeit with a much lower signal intensity than its paralogues Otx1 and Otx2. At later stages, four major expression sites are observed: the developing eye and epiphysis, the olfactory placodes and a broad epidermal domain in the dorsal part of the head. In the embryonic eye, the signal is first detected in the presumptive pigmented retina and slightly later in the adjacent outer layer of the neural retina, fated to photoreceptors. The comparison of this expression pattern with those of osteichthyan Otx genes suggests that a role in the specification of photoreceptors may correspond to a functional specialization of Otx5and Crx genes, fixed early in the gnathostome lineage, prior to the splitting of chondrichthyans and osteichthyans. In contrast, the roles played by ScOtx5 in the retinal pigmented epithelium or in the olfactory placodes may be fulfilled by different combinations of paralogous genes in other gnathostome taxa.

Betancur P, Simões-Costa M, Sauka-Spengler T, Bronner ME. 2014. Expression and function of transcription factor cMyb during cranial neural crest development. Mech Dev, 132 pp. 38-43. | Show Abstract | Read more

The transcription factor cMyb has well known functions in vertebrate hematopoiesis, but little was known about its distribution or function at early developmental stages. Here, we show that cMyb transcripts are present at the neural plate during gastrulation in chick embryos. cMyb expression then resolves to the cranial neural folds and is maintained in early migrating cranial neural crest cells during and after neurulation. Morpholino-mediated knock-down of cMyb reduces expression of Pax7 and Twist at the neural plate border, as well as reducing expression of neural crest specifier gene Slug/Snail2 and completely eliminating expression of Ets1. On the other hand, its loss results in abnormal maintenance of Zic1, but little or no effect on other neural crest specifier genes like FoxD3 or Sox9. These results place cMyb in a critical hierarchical position within the cranial neural crest cell gene regulatory network, likely directly inhibiting Zic1 and upstream of Ets1 and some, but not all, neural crest specifier genes.

Modrell MS, Hockman D, Uy B, Buckley D, Sauka-Spengler T, Bronner ME, Baker CVH. 2014. A fate-map for cranial sensory ganglia in the sea lamprey Developmental Biology, 385 (2), pp. 405-416. | Show Abstract | Read more

Cranial neurogenic placodes and the neural crest make essential contributions to key adult characteristics of all vertebrates, including the paired peripheral sense organs and craniofacial skeleton. Neurogenic placode development has been extensively characterized in representative jawed vertebrates (gnathostomes) but not in jawless fishes (agnathans). Here, we use in vivo lineage tracing with DiI, together with neuronal differentiation markers, to establish the first detailed fate-map for placode-derived sensory neurons in a jawless fish, the sea lamprey Petromyzon marinus, and to confirm that neural crest cells in the lamprey contribute to the cranial sensory ganglia. We also show that a pan-Pax3/7 antibody labels ophthalmic trigeminal (opV, profundal) placode-derived but not maxillomandibular trigeminal (mmV) placode-derived neurons, mirroring the expression of gnathostome Pax3 and suggesting that Pax3 (and its single Pax3/7 lamprey ortholog) is a pan-vertebrate marker for opV placode-derived neurons. Unexpectedly, however, our data reveal that mmV neuron precursors are located in two separate domains at neurula stages, with opV neuron precursors sandwiched between them. The different branches of the mmV nerve are not comparable between lampreys and gnatho-stomes, and spatial segregation of mmV neuron precursor territories may be a derived feature of lampreys. Nevertheless, maxillary and mandibular neurons are spatially segregated within gnathostome mmV ganglia, suggesting that a more detailed investigation of gnathostome mmV placode development would be worthwhile. Overall, however, our results highlight the conservation of cranial peripheral sensory nervous system development across vertebrates, yielding insight into ancestral vertebrate traits. © 2013 The Authors.

Simões-Costa M, Tan-Cabugao J, Antoshechkin I, Sauka-Spengler T, Bronner ME. 2014. Transcriptome analysis reveals novel players in the cranial neural crest gene regulatory network. Genome Res, 24 (2), pp. 281-290. | Show Abstract | Read more

The neural crest is an embryonic stem cell population that gives rise to a multitude of derivatives. In particular, the cranial neural crest (CNC) is unique in its ability to contribute to both facial skeleton and peripheral ganglia. To gain further insight into the molecular underpinnings that distinguish the CNC from other embryonic tissues, we have utilized a CNC-specific enhancer as a tool to isolate a pure, region-specific NC subpopulation for transcriptional profiling. The resulting data set reveals previously unknown transcription factors and signaling pathways that may influence the CNC's ability to migrate and/or differentiate into unique derivatives. To elaborate on the CNC gene regulatory network, we evaluated the effects of knocking down known neural plate border genes and early neural crest specifier genes on selected neural crest-enriched transcripts. The results suggest that ETS1 and SOX9 may act as pan-neural crest regulators of the migratory CNC. Taken together, our analysis provides unprecedented characterization of the migratory CNC transcriptome and identifies new links in the gene regulatory network responsible for development of this critical cell population.

Bassett AR, Azzam G, Wheatley L, Tibbit C, Rajakumar T, McGowan S, Stanger N, Ewels PA, Taylor S, Ponting CP et al. 2014. Understanding functional miRNA-target interactions in vivo by site-specific genome engineering. Nat Commun, 5 pp. 4640. | Show Abstract | Read more

MicroRNA (miRNA) target recognition is largely dictated by short 'seed' sequences, and single miRNAs therefore have the potential to regulate a large number of genes. Understanding the contribution of specific miRNA-target interactions to the regulation of biological processes in vivo remains challenging. Here we use transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technologies to interrogate the functional relevance of predicted miRNA response elements (MREs) to post-transcriptional silencing in zebrafish and Drosophila. We also demonstrate an effective strategy that uses CRISPR-mediated homology-directed repair with short oligonucleotide donors for the assessment of MRE activity in human cells. These methods facilitate analysis of the direct phenotypic consequences resulting from blocking specific miRNA-MRE interactions at any point during development.

Parker HJ, Sauka-Spengler T, Bronner M, Elgar G. 2014. A reporter assay in lamprey embryos reveals both functional conservation and elaboration of vertebrate enhancers. PLoS One, 9 (1), pp. e85492. | Show Abstract | Read more

The sea lamprey is an important model organism for investigating the evolutionary origins of vertebrates. As more vertebrate genome sequences are obtained, evolutionary developmental biologists are becoming increasingly able to identify putative gene regulatory elements across the breadth of the vertebrate taxa. The identification of these regions makes it possible to address how changes at the genomic level have led to changes in developmental gene regulatory networks and ultimately to the evolution of morphological diversity. Comparative genomics approaches using sea lamprey have already predicted a number of such regulatory elements in the lamprey genome. Functional characterisation of these sequences and other similar elements requires efficient reporter assays in lamprey. In this report, we describe the development of a transient transgenesis method for lamprey embryos. Focusing on conserved non-coding elements (CNEs), we use this method to investigate their functional conservation across the vertebrate subphylum. We find instances of both functional conservation and lineage-specific functional evolution of CNEs across vertebrates, emphasising the utility of functionally testing homologous CNEs in their host species.

Amemiya CT, Alföldi J, Lee AP, Fan S, Philippe H, Maccallum I, Braasch I, Manousaki T, Schneider I, Rohner N et al. 2013. The African coelacanth genome provides insights into tetrapod evolution. Nature, 496 (7445), pp. 311-316. | Show Abstract | Read more

The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.

Cited:

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Smith JJ, Kuraku S, Holt C, Sauka-Spengler T, Jiang N, Campbell MS, Yandell MD, Manousaki T, Meyer A, Bloom OE et al. 2013. Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution Nature Genetics, 45 (4), pp. 415-421. | Show Abstract | Read more

Lampreys are representatives of an ancient vertebrate lineage that diverged from our own ∼500 million years ago. By virtue of this deeply shared ancestry, the sea lamprey (P. marinus) genome is uniquely poised to provide insight into the ancestry of vertebrate genomes and the underlying principles of vertebrate biology. Here, we present the first lamprey whole-genome sequence and assembly. We note challenges faced owing to its high content of repetitive elements and GC bases, as well as the absence of broad-scale sequence information from closely related species. Analyses of the assembly indicate that two whole-genome duplications likely occurred before the divergence of ancestral lamprey and gnathostome lineages. Moreover, the results help define key evolutionary events within vertebrate lineages, including the origin of myelin-associated proteins and the development of appendages. The lamprey genome provides an important resource for reconstructing vertebrate origins and the evolutionary events that have shaped the genomes of extant organisms.

Simões-Costa MS, McKeown SJ, Tan-Cabugao J, Sauka-Spengler T, Bronner ME. 2012. Dynamic and differential regulation of stem cell factor FoxD3 in the neural crest is Encrypted in the genome. PLoS Genet, 8 (12), pp. e1003142. | Show Abstract | Read more

The critical stem cell transcription factor FoxD3 is expressed by the premigratory and migrating neural crest, an embryonic stem cell population that forms diverse derivatives. Despite its important role in development and stem cell biology, little is known about what mediates FoxD3 activity in these cells. We have uncovered two FoxD3 enhancers, NC1 and NC2, that drive reporter expression in spatially and temporally distinct manners. Whereas NC1 activity recapitulates initial FoxD3 expression in the cranial neural crest, NC2 activity recapitulates initial FoxD3 expression at vagal/trunk levels while appearing only later in migrating cranial crest. Detailed mutational analysis, in vivo chromatin immunoprecipitation, and morpholino knock-downs reveal that transcription factors Pax7 and Msx1/2 cooperate with the neural crest specifier gene, Ets1, to bind to the cranial NC1 regulatory element. However, at vagal/trunk levels, they function together with the neural plate border gene, Zic1, which directly binds to the NC2 enhancer. These results reveal dynamic and differential regulation of FoxD3 in distinct neural crest subpopulations, suggesting that heterogeneity is encrypted at the regulatory level. Isolation of neural crest enhancers not only allows establishment of direct regulatory connections underlying neural crest formation, but also provides valuable tools for tissue specific manipulation and investigation of neural crest cell identity in amniotes.

Hu N, Strobl-Mazzulla P, Sauka-Spengler T, Bronner ME. 2012. DNA methyltransferase3A as a molecular switch mediating the neural tube-to-neural crest fate transition. Genes Dev, 26 (21), pp. 2380-2385. | Show Abstract | Read more

Here, we explore whether silencing via promoter DNA methylation plays a role in neural versus neural crest cell lineage decisions. We show that DNA methyltransferase3A (DNMT3A) promotes neural crest specification by directly mediating repression of neural genes like Sox2 and Sox3. DNMT3A is expressed in the neural plate border, and its knockdown causes ectopic Sox2 and Sox3 expression at the expense of neural crest markers. In vivo chromatin immunoprecipitation of neural folds demonstrates that DNMT3A specifically associates with CpG islands in the Sox2 and Sox3 promoter regions, resulting in their repression by methylation. Thus, DNMT3A functions as a molecular switch, repressing neural to favor neural crest cell fate.

Betancur P, Sauka-Spengler T, Bronner M. 2011. A Sox10 enhancer element common to the otic placode and neural crest is activated by tissue-specific paralogs. Development, 138 (17), pp. 3689-3698. | Show Abstract | Read more

The otic placode, a specialized region of ectoderm, gives rise to components of the inner ear and shares many characteristics with the neural crest, including expression of the key transcription factor Sox10. Here, we show that in avian embryos, a highly conserved cranial neural crest enhancer, Sox10E2, also controls the onset of Sox10 expression in the otic placode. Interestingly, we show that different combinations of paralogous transcription factors (Sox8, Pea3 and cMyb versus Sox9, Ets1 and cMyb) are required to mediate Sox10E2 activity in the ear and neural crest, respectively. Mutating their binding motifs within Sox10E2 greatly reduces enhancer activity in the ear. Moreover, simultaneous knockdown of Sox8, Pea3 and cMyb eliminates not only the enhancer-driven reporter expression, but also the onset of endogenous Sox10 expression in the ear. Rescue experiments confirm that the specific combination of Myb together with Sox8 and Pea3 is responsible for the onset of Sox10 expression in the otic placode, as opposed to Myb plus Sox9 and Ets1 for neural crest Sox10 expression. Whereas SUMOylation of Sox8 is not required for the initial onset of Sox10 expression, it is necessary for later otic vesicle formation. This new role of Sox8, Pea3 and cMyb in controlling Sox10 expression via a common otic/neural crest enhancer suggests an evolutionarily conserved function for the combination of paralogous transcription factors in these tissues of distinct embryological origin.

Betancur P, Bronner-Fraser M, Sauka-Spengler T. 2010. Assembling neural crest regulatory circuits into a gene regulatory network. Annu Rev Cell Dev Biol, 26 (1), pp. 581-603. | Show Abstract | Read more

The neural crest is a multipotent stem cell&#x2013;like population that gives rise to a wide range of derivatives in the vertebrate embryo including elements of the craniofacial skeleton and peripheral nervous system as well as melanocytes. The neural crest forms in a series of regulatory steps that include induction and specification of the prospective neural crest territory&#x2013;neural plate border, specification of bona fide neural crest progenitors, and differentiation into diverse derivatives. These individual processes during neural crest ontogeny are controlled by regulatory circuits that can be assembled into a hierarchical gene regulatory network (GRN). Here we present an overview of the GRN that orchestrates the formation of cranial neural crest cells. Formulation of this network relies on information largely inferred from gene perturbation studies performed in several vertebrate model organisms. Our representation of the cranial neural crest GRN also includes information about direct regulatory interactions obtained from the cis-regulatory analyses performed to date, which increases the resolution of the architectural circuitry within the network.

Sauka-Spengler T, Bronner M. 2010. Snapshot: neural crest. Cell, 143 (3), pp. 486-486.e1. | Read more

Specifying neural crest cell from ‘scratch’

Neural crest (NC) is a unique multipotent embryonic cell population that differentiates into a plethora of diverse cell types, giving rise to structures as different as neurons and glia of peripheral nervous system, bone, cartilage and connective tissue elements of craniofacial skeleton and body’s pigmentation. Defects in neural crest patterning are some of the most common causes of birth anomalies, accounting for up to one-third of all congenital birth defects. Due to the unique multipotency, ...

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