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Neural crest cells exemplify cellular diversification from a multipotent progenitor population. However, the full sequence of early molecular choices orchestrating the emergence of neural crest heterogeneity from the embryonic ectoderm remains elusive. Gene-regulatory-networks (GRN) govern early development and cell specification toward definitive neural crest. Here, we combine ultradense single-cell transcriptomes with machine-learning and large-scale transcriptomic and epigenomic experimental validation of selected trajectories, to provide the general principles and highlight specific features of the GRN underlying neural crest fate diversification from induction to early migration stages using Xenopus frog embryos as a model. During gastrulation, a transient neural border zone state precedes the choice between neural crest and placodes which includes multiple converging gene programs. During neurulation, transcription factor connectome, and bifurcation analyses demonstrate the early emergence of neural crest fates at the neural plate stage, alongside an unbiased multipotent-like lineage persisting until epithelial-mesenchymal transition stage. We also decipher circuits driving cranial and vagal neural crest formation and provide a broadly applicable high-throughput validation strategy for investigating single-cell transcriptomes in vertebrate GRNs in development, evolution, and disease.

Frogs are an ecologically diverse and phylogenetically ancient group of anuran amphibians that include important vertebrate cell and developmental model systems, notably the genus Xenopus. Here we report a high-quality reference genome sequence for the western clawed frog, Xenopus tropicalis, along with draft chromosome-scale sequences of three distantly related emerging model frog species, Eleutherodactylus coqui, Engystomops pustulosus, and Hymenochirus boettgeri. Frog chromosomes have remained remarkably stable since the Mesozoic Era, with limited Robertsonian (i.e., arm-preserving) translocations and end-to-end fusions found among the smaller chromosomes. Conservation of synteny includes conservation of centromere locations, marked by centromeric tandem repeats associated with Cenp-a binding surrounded by pericentromeric LINE/L1 elements. This work explores the structure of chromosomes across frogs, using a dense meiotic linkage map for X. tropicalis and chromatin conformation capture (Hi-C) data for all species. Abundant satellite repeats occupy the unusually long (~20 megabase) terminal regions of each chromosome that coincide with high rates of recombination. Both embryonic and differentiated cells show reproducible associations of centromeric chromatin and of telomeres, reflecting a Rabl-like configuration. Our comparative analyses reveal 13 conserved ancestral anuran chromosomes from which contemporary frog genomes were constructed.

In many groups, sex chromosomes change frequently but the drivers of their rapid evolution are varied and often poorly characterized. With an aim of further understanding sex chromosome turnover, we investigated the polymorphic sex chromosomes of the Marsabit clawed frog, Xenopus borealis, using genomic data and a new chromosome-scale genome assembly. We confirmed previous findings that 54.1 Mb of chromosome 8L is sex-linked in animals from east Kenya and a laboratory strain, but most (or all) of this region is not sex-linked in natural populations from west Kenya. Previous work suggests possible degeneration of the Z chromosomes in the east population because many sex-linked transcripts of this female heterogametic population have female-biased expression, and we therefore expected this chromosome to not be present in the west population. In contrast, our simulations support a model where most or all of the sex-linked portion of the Z chromosome from the east acquired autosomal segregation in the west, and where much genetic variation specific to the large sex-linked portion of the W chromosome from the east is not present in the west. These recent changes are consistent with the hot-potato model, wherein sex chromosome turnover is favoured by natural selection if it purges a (minimally) degenerate sex-specific sex chromosome, but counterintuitively suggest natural selection failed to purge a Z chromosome that has signs of more advanced and possibly more ancient regulatory degeneration. These findings highlight complex evolutionary dynamics of young, rapidly evolving Xenopus sex chromosomes and set the stage for mechanistic work aimed at pinpointing additional sex-determining genes in this group.

Gene Ontology analyses of autism spectrum disorders (ASD) risk genes have repeatedly highlighted synaptic function and transcriptional regulation as key points of convergence. However, these analyses rely on incomplete knowledge of gene function across brain development. Here we leverage Xenopus tropicalis to study in vivo ten genes with the strongest statistical evidence for association with ASD. All genes are expressed in developing telencephalon at time points mapping to human mid-prenatal development, and mutations lead to an increase in the ratio of neural progenitor cells to maturing neurons, supporting previous in silico systems biological findings implicating cortical neurons in ASD vulnerability, but expanding the range of convergent functions to include neurogenesis. Systematic chemical screening identifies that estrogen, via Sonic hedgehog signaling, rescues this convergent phenotype in Xenopus and human models of brain development, suggesting a resilience factor that may mitigate a range of ASD genetic risks.

The Western clawed frog Xenopus tropicalis is a diploid model system for both frog genetics and developmental biology, complementary to the paleotetraploid X. laevis. Here we report a chromosome-scale assembly of the X. tropicalis genome, improving the previously published draft genome assembly through the use of new assembly algorithms, additional sequence data, and the addition of a dense genetic map. The improved genome enables the mapping of specific traits (e.g., the sex locus or Mendelian mutants) and the characterization of chromosome-scale synteny with other tetrapods. We also report an improved annotation of the genome that integrates deep transcriptome sequence from diverse tissues and stages. The exon-intron structures of these genes are highly conserved relative to both X. laevis and human, as are chromosomal linkages ("synteny") and local gene order. A network analysis of developmental gene expression will aid future studies.

RNA alternative polyadenylation contributes to the complexity of information transfer from genome to phenome, thus amplifying gene function. Here, we report the first X. tropicalis resource with 127,914 alternative polyadenylation (APA) sites derived from embryos and adults. Overall, APA networks play central roles in coordinating the maternal-zygotic transition (MZT) in embryos, sexual dimorphism in adults and longitudinal growth from embryos to adults. APA sites coordinate reprogramming in embryos before the MZT, but developmental events after the MZT due to zygotic genome activation. The APA transcriptomes of young adults are more variable than growing adults and male frog APA transcriptomes are more divergent than females. The APA profiles of young females were similar to embryos before the MZT. Enriched pathways in developing embryos were distinct across the MZT and noticeably segregated from adults. Briefly, our results suggest that the minimal functional units in genomes are alternative transcripts as opposed to genes.

Microtubule remodeling is critical for cellular and developmental processes underlying morphogenetic changes and for the formation of many subcellular structures. Katanins are conserved microtubule severing enzymes that are essential for spindle assembly, ciliogenesis, cell division, and cellular motility. We have recently shown that a related protein, Katanin-like 2 (KATNAL2), is similarly required for cytokinesis, cell cycle progression, and ciliogenesis in cultured mouse cells. However, its developmental expression pattern, localization, and in vivo role during organogenesis have yet to be characterized. Here, we used Xenopus embryos to reveal that Katnal2 (1) is expressed broadly in ciliated and neurogenic tissues throughout embryonic development; (2) is localized to basal bodies, ciliary axonemes, centrioles, and mitotic spindles; and (3) is required for ciliogenesis and brain development. Since human KATNAL2 is a risk gene for autism spectrum disorders, our functional data suggest that Xenopus may be a relevant system for understanding the relationship of mutations in this gene to autism and the underlying molecular mechanisms of pathogenesis.

Na+/H+ exchangers (NHEs) represent a highly conserved family of ion transporters that regulate pH homeostasis. NHEs as well as other proton transporters were previously linked to the regulation of the Wnt signaling pathway, cell polarity signaling, and mucociliary function. Furthermore, mutations in the gene SLC9A3 (encoding NHE3) were detected as additional risk factors for airway infections in cystic fibrosis patients. Here, we used the Xenopus embryonic mucociliary epidermis as well as human airway epithelial cells (HAECs) as models to investigate the functional roles of NHEs in mucociliary development and regeneration. In Xenopus embryos, NHEs 1-3 were expressed during epidermal development, and loss of NHE function impaired mucociliary clearance in tadpoles. Clearance defects were caused by reduced cilia formation, disrupted alignment of basal bodies in multiciliated cells (MCCs), and dysregulated mucociliary gene expression. These data also suggested that NHEs may contribute to the activation of Wnt signaling in mucociliary epithelia. In HAECs, pharmacological inhibition of NHE function also caused defective ciliation and regeneration in airway MCCs. Collectively, our data revealed a requirement for NHEs in vertebrate mucociliary epithelia and linked NHE activity to cilia formation and function in differentiating MCCs. Our results provide an entry point for the understanding of the contribution of NHEs to signaling, development, and pathogenesis in the human respiratory tract.

During vertebrate neurulation, the embryonic ectoderm is patterned into lineage progenitors for neural plate, neural crest, placodes and epidermis. Here, we use Xenopus laevis embryos to analyze the spatial and temporal transcriptome of distinct ectodermal domains in the course of neurulation, during the establishment of cell lineages. In order to define the transcriptome of small groups of cells from a single germ layer and to retain spatial information, dorsal and ventral ectoderm was subdivided along the anterior-posterior and medial-lateral axes by microdissections. Principal component analysis on the transcriptomes of these ectoderm fragments primarily identifies embryonic axes and temporal dynamics. This provides a genetic code to define positional information of any ectoderm sample along the anterior-posterior and dorsal-ventral axes directly from its transcriptome. In parallel, we use nonnegative matrix factorization to predict enhanced gene expression maps onto early and mid-neurula embryos, and specific signatures for each ectoderm area. The clustering of spatial and temporal datasets allowed detection of multiple biologically relevant groups (e.g., Wnt signaling, neural crest development, sensory placode specification, ciliogenesis, germ layer specification). We provide an interactive network interface, EctoMap, for exploring synexpression relationships among genes expressed in the neurula, and suggest several strategies to use this comprehensive dataset to address questions in developmental biology as well as stem cell or cancer research.

The spacing of hair in mammals and feathers in birds is one of the most apparent morphological features of the skin. This pattern arises when uniform fields of progenitor cells diversify their molecular fate while adopting higher-order structure. Using the nascent skin of the developing chicken embryo as a model system, we find that morphological and molecular symmetries are simultaneously broken by an emergent process of cellular self-organization. The key initiators of heterogeneity are dermal progenitors, which spontaneously aggregate through contractility-driven cellular pulling. Concurrently, this dermal cell aggregation triggers the mechanosensitive activation of β-catenin in adjacent epidermal cells, initiating the follicle gene expression program. Taken together, this mechanism provides a means of integrating mechanical and molecular perspectives of organ formation.

The canonical Wnt/β-catenin signaling pathway plays multiple roles during Xenopus gastrulation, including posteriorization of the neural plate, patterning of the mesoderm, and induction of the neural crest. Wnt signaling stabilizes β-catenin, which then activates target genes. However, few targets of this signaling pathway that mediate early developmental processes are known. Here we sought to identify transcriptional targets of the Wnt/β-catenin signaling pathway using a genome-wide approach. We selected putative targets using the criteria of reduced expression upon zygotic Wnt knockdown, β-catenin binding within 50kb of the gene, and expression in tissues that receive Wnt signaling. Using these criteria, we found 21 novel direct transcriptional targets of Wnt/β-catenin signaling during gastrulation and in addition have identified putative regulatory elements for further characterization in future studies.

The dorsal ventral axis of vertebrates requires high BMP activity for ventral development and inhibition of BMP activity for dorsal development. Presumptive dorsal regions of the embryo are protected from the ventralizing activity of BMPs by the secretion of BMP antagonists from the mesoderm. Noggin, one such antagonist, binds BMP ligands and prevents them from binding their receptors, however, a unique role for Noggin in amphibian development has remained unclear. Previously, we used zinc-finger nucleases to mutagenize the noggin locus in Xenopus tropicalis. Here, we report on the phenotype of noggin mutant frogs as a result of breeding null mutations to homozygosity. Early homozygous noggin mutant embryos are indistinguishable from wildtype siblings, with normal neural induction and neural tube closure. However, in late tadpole stages mutants present severe ventral craniofacial defects, notably a fusion of Meckel's cartilage to the palatoquadrate cartilage. Consistent with a noggin loss-of-function, mutants show expansions of BMP target gene expression and the mutant phenotype can be rescued with transient BMP inhibition. These results demonstrate that in amphibians, Noggin is dispensable for early embryonic patterning but is critical for cranial skeletogenesis.

To define a complete catalog of the genes that are activated during mouse sclerotome formation, we sequenced RNA from embryonic mouse tissue directed to form sclerotome in culture. In addition to well-known early markers of sclerotome, such as Pax1, Pax9, and the Bapx2/Nkx3-2 homolog Nkx3-1, the long-noncoding RNA PEAT (Pax1 enhancer antisense transcript) was induced in sclerotome-directed samples. Strikingly, PEAT is located just upstream of the Pax1 gene. Using CRISPR/Cas9, we generated a mouse line bearing a complete deletion of the PEAT-transcribed unit. RNA-seq on PEAT mutant embryos showed that loss of PEAT modestly increases bone morphogenetic protein target gene expression and also elevates the expression of a large subset of ribosomal protein mRNAs.

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of 'fossil' transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

Upon cell cycle exit, centriole-to-basal body transition facilitates cilia formation. The centriolar protein Cp110 is a regulator of this process and cilia inhibitor, but its positive roles in ciliogenesis remain poorly understood. Using Xenopus we show that Cp110 inhibits cilia formation at high levels, while optimal levels promote ciliogenesis. Cp110 localizes to cilia-forming basal bodies and rootlets, and is required for ciliary adhesion complexes that facilitate Actin interactions. The opposing roles of Cp110 in ciliation are generated in part by coiled-coil domains that mediate preferential binding to centrioles over rootlets. Because of its dual role in ciliogenesis, Cp110 levels must be precisely controlled. In multiciliated cells, this is achieved by both transcriptional and post-transcriptional regulation through ciliary transcription factors and microRNAs, which activate and repress cp110 to produce optimal Cp110 levels during ciliogenesis. Our data provide novel insights into how Cp110 and its regulation contribute to development and cell function.

Construction of next-generation sequencing (NGS) libraries involves RNA manipulation, which often creates noisy, biased, and artifactual data that contribute to errors in transcriptome analysis. In this study, a total of 19 whole transcriptome termini site sequencing (WTTS-seq) and seven RNA sequencing (RNA-seq) libraries were prepared from Xenopus tropicalis adult and embryo samples to determine the most effective library preparation method to maximize transcriptomics investigation. We strongly suggest that appropriate primers/adaptors are designed to inhibit amplification detours and that PCR overamplification is minimized to maximize transcriptome coverage. Furthermore, genome annotation must be improved so that missing data can be recovered. In addition, a complete understanding of sequencing platforms is critical to limit the formation of false-positive results. Technically, the WTTS-seq method enriches both poly(A)+ RNA and complementary DNA, adds 5'- and 3'-adaptors in one step, pursues strand sequencing and mapping, and profiles both gene expression and alternative polyadenylation (APA). Although RNA-seq is cost prohibitive, tends to produce false-positive results, and fails to detect APA diversity and dynamics, its combination with WTTS-seq is necessary to validate transcriptome-wide APA.

Proton pump inhibitors (PPIs), which target gastric H(+)/K(+)ATPase (ATP4), are among the most commonly prescribed drugs. PPIs are used to treat ulcers and as a preventative measure against gastroesophageal reflux disease in hospitalized patients. PPI treatment correlates with an increased risk for airway infections, i.e. community- and hospital-acquired pneumonia. The cause for this correlation, however, remains elusive. The Xenopus embryonic epidermis is increasingly being used as a model to study airway-like mucociliary epithelia. Here we use this model to address how ATP4 inhibition may affect epithelial function in human airways. We demonstrate that atp4a knockdown interfered with the generation of cilia-driven extracellular fluid flow. ATP4a and canonical Wnt signaling were required in the epidermis for expression of foxj1, a transcriptional regulator of motile ciliogenesis. The ATP4/Wnt module activated foxj1 downstream of ciliated cell fate specification. In multiciliated cells (MCCs) of the epidermis, ATP4a was also necessary for normal myb expression, apical actin formation, basal body docking and alignment of basal bodies. Furthermore, ATP4-dependent Wnt/β-catenin signaling in the epidermis was a prerequisite for foxa1-mediated specification of small secretory cells (SSCs). SSCs release serotonin and other substances into the medium, and thereby regulate ciliary beating in MCCs and protect the epithelium against infection. Pharmacological inhibition of ATP4 in the mature mucociliary epithelium also caused a loss of MCCs and led to impaired mucociliary clearance. These data strongly suggest that PPI-associated pneumonia in human patients might, at least in part, be linked to dysfunction of mucociliary epithelia of the airways.

During gastrulation and neurulation, foxj1 expression requires ATP4a-dependent Wnt/β-catenin signaling for ciliation of the gastrocoel roof plate (Walentek et al. Cell Rep. 1 (2012) 516-527.) and the mucociliary epidermis (Walentek et al. Dev. Biol. (2015)) of Xenopus laevis embryos. These data suggested that ATP4a and Wnt/β-catenin signaling regulate foxj1 throughout Xenopus development. Here we analyzed whether foxj1 expression was also ATP4a-dependent in other ciliated tissues of the developing Xenopus embryo and tadpole. We found that in the floor plate of the neural tube ATP4a-dependent canonical Wnt signaling was required for foxj1 expression, downstream of or in parallel to Hedgehog signaling. In the developing tadpole brain, ATP4-function was a prerequisite for the establishment of cerebrospinal fluid flow. Furthermore, we describe foxj1 expression and the presence of multiciliated cells in the developing tadpole gastrointestinal tract. Our work argues for a general requirement of ATP4-dependent Wnt/β-catenin signaling for foxj1 expression and motile ciliogenesis throughout Xenopus development.

Whole transcriptome analysis plays an essential role in deciphering genome structure and function, identifying genetic networks underlying cellular, physiological, biochemical and biological systems and establishing molecular biomarkers that respond to diseases, pathogens and environmental challenges. Here, we review transcriptome analysis methods and technologies that have been used to conduct whole transcriptome shotgun sequencing or whole transcriptome tag/target sequencing analyses. We focus on how adaptors/linkers are added to both 5' and 3' ends of mRNA molecules for cloning or PCR amplification before sequencing. Challenges and potential solutions are also discussed. In brief, next generation sequencing platforms have accelerated releases of the large amounts of gene expression data. It is now time for the genome research community to assemble whole transcriptomes of all species and collect signature targets for each gene/transcript, and thus use known genes/transcripts to determine known transcriptomes directly in the near future.

Alternative splicing is pervasive in vertebrates, yet little is known about most isoforms or their regulation. transformer-2b (tra2b) encodes a splicing regulator whose endogenous function is poorly understood. Tra2b knockdown in Xenopus results in embryos with multiple defects, including defective somitogenesis. Using RNA sequencing, we identify 142 splice changes (mostly intron retention and exon skipping), 89% of which are not in current annotations. A previously undescribed isoform of wnt11b retains the last intron, resulting in a truncated ligand (Wnt11b-short). We show that this isoform acts as a dominant-negative ligand in cardiac gene induction and pronephric tubule formation. To determine the contribution of Wnt11b-short to the tra2b phenotype, we induce retention of intron 4 in wnt11b, which recapitulates the failure to form somites but not other tra2b morphant defects. This alternative splicing of a Wnt ligand adds intricacy to a complex signaling pathway and highlights intron retention as a regulatory mechanism.

The mir-34/449 family consists of six homologous miRNAs at three genomic loci. Redundancy of miR-34/449 miRNAs and their dominant expression in multiciliated epithelia suggest a functional significance in ciliogenesis. Here we report that mice deficient for all miR-34/449 miRNAs exhibited postnatal mortality, infertility and strong respiratory dysfunction caused by defective mucociliary clearance. In both mouse and Xenopus, miR-34/449-deficient multiciliated cells (MCCs) exhibited a significant decrease in cilia length and number, due to defective basal body maturation and apical docking. The effect of miR-34/449 on ciliogenesis was mediated, at least in part, by post-transcriptional repression of Cp110, a centriolar protein suppressing cilia assembly. Consistent with this, cp110 knockdown in miR-34/449-deficient MCCs restored ciliogenesis by rescuing basal body maturation and docking. Altogether, our findings elucidate conserved cellular and molecular mechanisms through which miR-34/449 regulate motile ciliogenesis.

A forward genetic screen of N-ethyl-N-nitrosourea mutagenized Xenopus tropicalis has identified an inner ear mutant named eclipse (ecl). Mutants developed enlarged otic vesicles and various defects of otoconia development; they also showed abnormal circular and inverted swimming patterns. Positional cloning identified specificity protein 8 (sp8), which was previously found to regulate limb and brain development. Two different loss-of-function approaches using transcription activator-like effector nucleases and morpholino oligonucleotides confirmed that the ecl mutant phenotype is caused by down-regulation of sp8. Depletion of sp8 resulted in otic dysmorphogenesis, such as uncompartmentalized and enlarged otic vesicles, epithelial dilation with abnormal sensory end organs. When overexpressed, sp8 was sufficient to induce ectopic otic vesicles possessing sensory hair cells, neurofilament innervation in a thickened sensory epithelium, and otoconia, all of which are found in the endogenous otic vesicle. We propose that sp8 is an important factor for initiation and elaboration of inner ear development.

Neural crest development is orchestrated by a complex and still poorly understood gene regulatory network. Premigratory neural crest is induced at the lateral border of the neural plate by the combined action of signaling molecules and transcription factors such as AP2, Gbx2, Pax3 and Zic1. Among them, Pax3 and Zic1 are both necessary and sufficient to trigger a complete neural crest developmental program. However, their gene targets in the neural crest regulatory network remain unknown. Here, through a transcriptome analysis of frog microdissected neural border, we identified an extended gene signature for the premigratory neural crest, and we defined novel potential members of the regulatory network. This signature includes 34 novel genes, as well as 44 known genes expressed at the neural border. Using another microarray analysis which combined Pax3 and Zic1 gain-of-function and protein translation blockade, we uncovered 25 Pax3 and Zic1 direct targets within this signature. We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. In addition, they may modulate the transcriptional output of multiple signaling pathways involved in neural crest development (Wnt, Retinoic Acid) through the induction of key pathway regulators (Axin2 and Cyp26c1). We also found that Pax3 could maintain its own expression through a positive autoregulatory feedback loop. These hierarchical inductions, feedback loops, and pathway modulations provide novel tools to understand the neural crest induction network.

When compared to single mutants for Follistatin or Noggin, we find that double mutants display a dramatic further reduction in trunk cartilage formation, particularly in the vertebral bodies and proximal ribs. Consistent with these observations, expression of the early sclerotome markers Pax1 and Uncx is diminished in Noggin;Follistatin compound mutants. In contrast, Sim1 expression expands medially in double mutants. As the onset of Follistatin expression coincides with sclerotome specification, we argue that the effect of Follistatin occurs after sclerotome induction. We hypothesize that Follistatin aids in maintaining proper somite size, and consequently sclerotome progenitor numbers, by preventing paraxial mesoderm from adopting an intermediate/lateral plate mesodermal fate in the Noggin-deficient state.

WNT signaling is critical in most aspects of skeletal development and homeostasis, and antagonists of WNT signaling are emerging as key regulatory proteins with great promise as therapeutic agents for bone disorders. Here we show that Sost and its paralog Sostdc1 emerged through ancestral genome duplication and their expression patterns have diverged to delineate non-overlapping domains in most organ systems including musculoskeletal, cardiovascular, nervous, digestive, reproductive and respiratory. In the developing limb, Sost and Sostdc1 display dynamic expression patterns with Sost being restricted to the distal ectoderm and Sostdc1 to the proximal ectoderm and the mesenchyme. While Sostdc1(-/-) mice lack any obvious limb or skeletal defects, Sost(-/-) mice recapitulate the hand defects described for Sclerosteosis patients. However, elevated WNT signaling in Sost(-/-); Sostdc1(-/-) mice causes misregulation of SHH signaling, ectopic activation of Sox9 in the digit 1 field and preaxial polydactyly in a Gli1- and Gli3-dependent manner. In addition, we show that the syndactyly documented in Sclerosteosis is present in both Sost(-/-) and Sost(-/-); Sostdc1(-/-) mice, and is driven by misregulation of Fgf8 in the AER, a region lacking Sost and Sostdc1 expression. This study highlights the complexity of WNT signaling in skeletal biology and disease and emphasizes how redundant mechanism and non-cell autonomous effects can synergize to unveil new intricate phenotypes caused by elevated WNT signaling.

Identifying gene regulatory elements and their target genes in vertebrates remains a significant challenge. It is now recognized that transcriptional regulatory sequences are critical in orchestrating dynamic controls of tissue-specific gene expression during vertebrate development and in adult tissues, and that these elements can be positioned at great distances in relation to the promoters of the genes they control. While significant progress has been made in mapping DNA binding regions by combining chromatin immunoprecipitation and next generation sequencing, functional validation remains a limiting step in improving our ability to correlate in silico predictions with biological function. We recently developed a computational method that synergistically combines genome-wide gene-expression profiling, vertebrate genome comparisons, and transcription factor binding-site analysis to predict tissue-specific enhancers in the human genome. We applied this method to 270 genes highly expressed in skeletal muscle and predicted 190 putative cis-regulatory modules. Furthermore, we optimized Tol2 transgenic constructs in Xenopus laevis to interrogate 20 of these elements for their ability to function as skeletal muscle-specific transcriptional enhancers during embryonic development. We found 45% of these elements expressed only in the fast muscle fibers that are oriented in highly organized chevrons in the Xenopus laevis tadpole. Transcription factor binding site analysis identified >2 Mef2/MyoD sites within ~200 bp regions in 6 of the validated enhancers, and systematic mutagenesis of these sites revealed that they are critical for the enhancer function. The data described herein introduces a new reporter system suitable for interrogating tissue-specific cis-regulatory elements which allows monitoring of enhancer activity in real time, throughout early stages of embryonic development, in Xenopus.

HAPPY mapping was designed to pursue the analysis of approximately random HAPloid DNA breakage samples using the PolYmerase chain reaction for mapping genomes. In the present study, we improved the method and integrated two other molecular techniques into the process: whole genome amplification and the Sequenom SNP (single nucleotide polymorphism) genotyping assay in order to facilitate whole genome mapping of X. tropicalis. The former technique amplified enough DNA materials to genotype a large number of markers, while the latter allowed for relatively high throughput marker genotyping with multiplex assays on the HAPPY lines. A total of 58 X. tropicalis genes were genotyped on an initial panel of 383 HAPPY lines, which contributed to formation of a working panel of 146 lines. Further genotyping of 29 markers on the working panel led to construction of a HAPPY map for the X. tropicalis genome. We believe that our improved HAPPY method described in the present study has paved the way for the community to map different genomes with a simple, but powerful approach.

HAPPY mapping was designed to pursue the analysis of approximately random HAPloid DNA breakage samples using the PolYmerase chain reaction for mapping genomes. In the present study, we improved the method and integrated two other molecular techniques into the process: whole genome amplification and the Sequenom SNP (single nucleotide polymorphism) genotyping assay in order to facilitate whole genome mapping of X. tropicalis. The former technique amplified enough DNA materials to genotype a large number of markers, while the latter allowed for relatively high throughput marker genotyping with multiplex assays on the HAPPY lines. A total of 58 X. tropicalis genes were genotyped on an initial panel of 383 HAPPY lines, which contributed to formation of a working panel of 146 lines. Further genotyping of 29 markers on the working panel led to construction of a HAPPY map for the X. tropicalis genome. We believe that our improved HAPPY method described in the present study has paved the way for the community to map different genomes with a simple, but powerful approach.

During skeletal development and regeneration, bone-forming osteoblasts respond to high metabolic demand by active expansion of their rough endoplasmic reticulum (rER) and increased synthesis of type I collagen, the predominant bone matrix protein. However, the molecular mechanisms that orchestrate this response are not well understood. We show that insertional mutagenesis of the previously uncharacterized osteopotentia (Opt) gene disrupts osteoblast function and causes catastrophic defects in postnatal skeletal development. Opt encodes a widely expressed rER-localized integral membrane protein containing a conserved SUN (Sad1/Unc-84 homology) domain. Mice lacking Opt develop acute onset skeletal defects that include impaired bone formation and spontaneous fractures. These defects result in part from a cell-autonomous failure of osteoblast maturation and a posttranscriptional decline in type I collagen synthesis, which is concordant with minimal rER expansion. By identifying Opt as a crucial regulator of bone formation in the mouse, our results uncover a novel rER-mediated control point in osteoblast function and implicate human Opt as a candidate gene for brittle bone disorders.

During the last three decades, both genome mapping and sequencing methods have advanced significantly to provide a foundation for scientists to understand genome structures and functions in many species. Generally speaking, genome mapping relies on genome sequencing to provide basic materials, such as DNA probes and markers for their localizations, thus constructing the maps. On the other hand, genome sequencing often requires a high-resolution map as a skeleton for whole genome assembly. However, both genome mapping and sequencing have never come together in one pipeline. After reviewing mapping and next-generation sequencing methods, we would like to share our thoughts with the genome community on how to combine the HAPPY mapping technique with the new-generation sequencing, thus integrating two systems into one pipeline, called HAPPY pipeline. The pipeline starts with preparation of a HAPPY panel, followed by multiple displacement amplification for producing a relatively large quantity of DNA. Instead of conventional marker genotyping, the amplified panel DNA samples are subject to new-generation sequencing with barcode method, which allows us to determine the presence/absence of a sequence contig as a traditional marker in the HAPPY panel. Statistical analysis will then be performed to infer how close or how far away from each other these contigs are within a genome and order the whole genome sequence assembly as well. We believe that such a universal approach will play an important role in genome sequencing, mapping, and assembly of many species; thus advancing genome science and its applications in biomedicine and agriculture.

BackgroundNon-sequence gene data (images, literature, etc.) can be found in many different public databases. Access to these data is mostly by text based methods using gene names; however, gene annotation is neither complete, nor fully systematic between organisms, and is also not generally stable over time. This provides some challenges for text based access, especially for cross-species searches. We propose a method for non-sequence data retrieval based on sequence similarity, which removes dependence on annotation and text searches. This work was motivated by the need to provide better access to large numbers of in situ images, and the observation that such image data were usually associated with a specific gene sequence. Sequence similarity searches are found in existing gene oriented databases, but mostly give indirect access to non-sequence data via navigational links.ResultsThree applications were built to explore the proposed method: accessing image data, literature and gene names. Searches are initiated with the sequence of the user's gene of interest, which is searched against a database of sequences associated with the target data. The matching (non-sequence) target data are returned directly to the user's browser, organised by sequence similarity. The method worked well for the intended application in image data management. Comparison with text based searches of the image data set showed the accuracy of the method. Applied to literature searches it facilitated retrieval of mostly high relevance references. Applied to gene name data it provided a useful analysis of name variation of related genes within and between species.ConclusionThis method makes a powerful and useful addition to existing methods for searching gene data based on text retrieval or curated gene lists. In particular the method facilitates cross-species comparisons, and enables the handling of novel or otherwise un-annotated genes. Applications using the method are quick and easy to build, and the data require little maintenance. This approach largely circumvents the need for annotation, which can be a major obstacle to the development of genomic scale data resources.

The African clawed frog Xenopus laevis has been instrumental to investigations of both development and cell biology, but the utility of this model organism for genetic and proteomic studies is limited by its long generation time and unsequenced pseudotetraploid genome. Xenopus tropicalis, which is a small, faster-breeding relative of X. laevis, has recently been adopted for research in developmental genetics and functional genomics, and has been chosen for genome sequencing. We show that X. tropicalis egg extracts reconstitute the fundamental cell cycle events of nuclear formation and bipolar spindle assembly around exogenously added sperm nuclei. Interestingly, X. tropicalis spindles were approximately 30% shorter than X. laevis spindles, and mixing experiments revealed a dynamic, dose-dependent regulation of spindle size by cytoplasmic factors. Measurements of microtubule dynamics revealed that microtubules polymerized slower in X. tropicalis extracts compared to X. laevis, but that this difference is unlikely to account for differences in spindle size. Thus, in addition to expanding the range of developmental and cell biological experiments, the use of X. tropicalis provides novel insight into the complex mechanisms that govern spindle morphogenesis.