The advent of next-generation sequencing (NGS) has revolutionized genomic and transcriptomic approaches to biology. These new sequencing tools are also valuable for the discovery, validation and assessment of genetic markers in populations. Here we review and discuss best practices for several NGS methods for genome-wide genetic marker development and genotyping that use restriction enzyme digestion of target genomes to reduce the complexity of the target. These new methods -- which include reduced-representation sequencing using reduced-representation libraries (RRLs) or complexity reduction of polymorphic sequences (CRoPS), restriction-site-associated DNA sequencing (RAD-seq) and low coverage genotyping -- are applicable to both model organisms with high-quality reference genome sequences and, excitingly, to non-model species with no existing genomic data.

Mammalian neural stem cells generate transit amplifying progenitors that expand the neuronal population, but these type of progenitors have not been studied in Drosophila. The Drosophila larval brain contains approximately 100 neural stem cells (neuroblasts) per brain lobe, which are thought to bud off smaller ganglion mother cells (GMCs) that each produce two post-mitotic neurons. Here, we use molecular markers and clonal analysis to identify a novel neuroblast cell lineage containing "transit amplifying GMCs" (TA-GMCs). TA-GMCs differ from canonical GMCs in several ways: each TA-GMC has nuclear Deadpan, cytoplasmic Prospero, forms Prospero crescents at mitosis, and generates up to 10 neurons; canonical GMCs lack Deadpan, have nuclear Prospero, lack Prospero crescents at mitosis, and generate two neurons. We conclude that there are at least two types of neuroblast lineages: a Type I lineage where GMCs generate two neurons, and a type II lineage where TA-GMCs have longer lineages. Type II lineages allow more neurons to be produced faster than Type I lineages, which may be advantageous in a rapidly developing organism like Drosophila.

BackgroundThe choice of a stem cell to divide symmetrically or asymmetrically has profound consequences for development and disease. Unregulated symmetric division promotes tumor formation, whereas inappropriate asymmetric division affects organ morphogenesis. Despite its importance, little is known about how spindle positioning is regulated. In some tissues cell fate appears to dictate the type of cell division, whereas in other tissues it is thought that stochastic variation in spindle position dictates subsequent sibling cell fate.ResultsHere we investigate the relationship between neural progenitor identity and spindle positioning in the Drosophila optic lobe. We use molecular markers and live imaging to show that there are two populations of progenitors in the optic lobe: symmetrically dividing neuroepithelial cells and asymmetrically dividing neuroblasts. We use genetically marked single cell clones to show that neuroepithelial cells give rise to neuroblasts. To determine if a change in spindle orientation can trigger a neuroepithelial to neuroblast transition, we force neuroepithelial cells to divide along their apical/basal axis by misexpressing Inscuteable. We find that this does not induce neuroblasts, nor does it promote premature neuronal differentiation.ConclusionWe show that symmetrically dividing neuroepithelial cells give rise to asymmetrically dividing neuroblasts in the optic lobe, and that regulation of spindle orientation and division symmetry is a consequence of cell type specification, rather than a mechanism for generating cell type diversity.

Next-generation sequencing has made it possible to begin asking questions about the process of divergence at the level of the genome. For example, recently, there has been a debate around the role of 'genomic islands of divergence' (i.e. blocks of outlier loci) in facilitating the process of speciation-with-gene-flow. The Swainson's thrush, Catharus ustulatus, is a migratory songbird with two genetically distinct subspecies that differ in a number of traits known to be involved in reproductive isolation in birds (plumage coloration, song and migratory behaviour), despite contemporary gene flow along a secondary contact zone. Here, we use RAD-PE sequencing to test emerging hypotheses about the process of divergence at the level of the genome and identify genes and gene regions involved in differentiation in this migratory songbird. Our analyses revealed distinct genomic islands on 15 of the 23 chromosomes and an accelerated rate of divergence on the Z chromosome, one of the avian sex chromosomes. Further, an analysis of loci linked to traits known to be involved in reproductive isolation in songbirds showed that genes linked to migration are significantly more differentiated than expected by chance, but that these genes lie primarily outside the genomic islands. Overall, our analysis supports the idea that genes linked to migration play an important role in divergence in migratory songbirds, but we find no compelling evidence that the observed genomic islands are facilitating adaptive divergence in migratory behaviour.

Tissue homeostasis depends on the ability of stem cells to properly regulate self-renewal versus differentiation. Drosophila neural stem cells (neuroblasts) are a model system to study self-renewal and differentiation. Recent work has identified two types of larval neuroblasts that have different self-renewal/differentiation properties. Type I neuroblasts bud off a series of small basal daughter cells (ganglion mother cells) that each generate two neurons. Type II neuroblasts bud off small basal daughter cells called intermediate progenitors (INPs), with each INP generating 6 to 12 neurons. Type I neuroblasts and INPs have nuclear Asense and cytoplasmic Prospero, whereas type II neuroblasts lack both these transcription factors. Here we test whether Prospero distinguishes type I/II neuroblast identity or proliferation profile, using several newly characterized Gal4 lines. We misexpress prospero using the 19H09-Gal4 line (expressed in type II neuroblasts but no adjacent type I neuroblasts) or 9D11-Gal4 line (expressed in INPs but not type II neuroblasts). We find that differential prospero expression does not distinguish type I and type II neuroblast identities, but Prospero regulates proliferation in both type I and type II neuroblast lineages. In addition, we use 9D11 lineage tracing to show that type II lineages generate both small-field and large-field neurons within the adult central complex, a brain region required for locomotion, flight, and visual pattern memory.

The Drosophila Activin-like ligands Activin-β and Dawdle control several aspects of neuronal morphogenesis, including mushroom body remodeling, dorsal neuron morphogenesis and motoneuron axon guidance. Here we show that the same two ligands act redundantly through the Activin receptor Babo and its transcriptional mediator Smad2 (Smox), to regulate neuroblast numbers and proliferation rates in the developing larval brain. Blocking this pathway results in the development of larvae with small brains and aberrant photoreceptor axon targeting, and restoring babo function in neuroblasts rescued these mutant phenotypes. These results suggest that the Activin signaling pathway is required for producing the proper number of neurons to enable normal connection of incoming photoreceptor axons to their targets. Furthermore, as the Activin pathway plays a key role in regulating propagation of mouse and human embryonic stem cells, our observation that it also regulates neuroblast numbers and proliferation in Drosophilasuggests that involvement of Activins in controlling stem cell propagation may be a common regulatory feature of this family of TGF-β-type ligands.

The ability of animals to carry out their normal behavioral repertoires requires exquisitely precise matching between specific motoneuron subtypes and the muscles they innervate. However, the molecular mechanisms that regulate motoneuron subtype specification remain unclear. Here, we use individually identified zebrafish primary motoneurons to describe a novel role for Nkx6 and Islet1 proteins in the specification of vertebrate motoneuron subtypes. We show that zebrafish primary motoneurons express two related Nkx6 transcription factors. In the absence of both Nkx6 proteins, the CaP motoneuron subtype develops normally, whereas the MiP motoneuron subtype develops a more interneuron-like morphology. In the absence of Nkx6 function, MiPs exhibit normal early expression of islet1, which is required for motoneuron formation; however, they fail to maintain islet1 expression. Misexpression of islet1 RNA can compensate for loss of Nkx6 function, providing evidence that Islet1 acts downstream of Nkx6. We suggest that Nkx6 proteins regulate MiP development at least in part by maintaining the islet1 expression that is required both to promote the MiP subtype and to suppress interneuron development.

Top predators are disappearing worldwide, significantly changing ecosystems that depend on top-down regulation. Conflict with humans remains the primary roadblock for large carnivore conservation, but for the eastern wolf (Canis lycaon), disagreement over its evolutionary origins presents a significant barrier to conservation in Canada and has impeded protection for grey wolves (Canis lupus) in the USA. Here, we use 127 235 single-nucleotide polymorphisms (SNPs) identified from restriction-site associated DNA sequencing (RAD-seq) of wolves and coyotes, in combination with genomic simulations, to test hypotheses of hybrid origins of Canis types in eastern North America. A principal components analysis revealed no evidence to support eastern wolves, or any other Canis type, as the product of grey wolf × western coyote hybridization. In contrast, simulations that included eastern wolves as a distinct taxon clarified the hybrid origins of Great Lakes-boreal wolves and eastern coyotes. Our results support the eastern wolf as a distinct genomic cluster in North America and help resolve hybrid origins of Great Lakes wolves and eastern coyotes. The data provide timely information that will shed new light on the debate over wolf conservation in eastern North America.