Selective expansion of motor cortical projections in the evolution of vocal novelty.

Deciphering how cortical architecture evolves to drive behavioral innovations is a long-standing challenge in neuroscience and evolutionary biology. Here, we leverage a striking behavioral novelty in the Alston's singing mouse (), compared to the laboratory mouse (), to quantitatively test models of motor cortical evolution. We used bulk tracing, serial two-photon tomography, and high-throughput DNA sequencing of over 76,000 barcoded neurons to discover a specific and substantial expansion (200%) of orofacial motor cortical (OMC) projections to the auditory cortical region (AudR) and the midbrain periaqueductal gray (PAG), both implicated in vocal behaviors.

Massive multiplexing of spatially resolved single neuron projections with axonal BARseq.

Neurons in the cortex are heterogeneous, sending diverse axonal projections to multiple brain regions. Unraveling the logic of these projections requires single-neuron resolution. Although a growing number of techniques have enabled high-throughput reconstruction, these techniques are typically limited to dozens or at most hundreds of neurons per brain, requiring that statistical analyses combine data from different specimens.

Spontaneously regenerative corticospinal neurons in mice.

The spinal cord receives inputs from the cortex via corticospinal neurons (CSNs). While predominantly a contralateral projection, a less-investigated minority of its axons terminate in the ipsilateral spinal cord. We analyzed the spatial and molecular properties of these ipsilateral axons and their post-synaptic targets in mice and found they project primarily to the ventral horn, including directly to motor neurons.

Single basolateral amygdala neurons in macaques exhibit distinct connectional motifs with frontal cortex.

Basolateral amygdala (BLA) projects widely across the macaque frontal cortex, and amygdalo-frontal projections are critical for appropriate emotional responding and decision making. While it is appreciated that single BLA neurons branch and project to multiple areas in frontal cortex, the organization and frequency of this branching has yet to be fully characterized. Here, we determined the projection patterns of more than 3,000 macaque BLA neurons.

Massive Multiplexing of Spatially Resolved Single Neuron Projections with Axonal BARseq.

Neurons in the cortex are heterogenous, sending diverse axonal projections to multiple brain regions. Unraveling the logic of these projections requires single-neuron resolution. Although a growing number of techniques have enabled high-throughput reconstruction, these techniques are typically limited to dozens or at most hundreds of neurons per brain, requiring that statistical analyses combine data from different specimens.

High-throughput sequencing of macaque basolateral amygdala projections reveals dissociable connectional motifs with frontal cortex.

The basolateral amygdala (BLA) projects widely across the macaque frontal cortex, and amygdalo-frontal projections are critical for optimal emotional responding and decision-making. Yet, little is known about the single-neuron architecture of these projections: namely, whether single BLA neurons project to multiple parts of the frontal cortex. Here, we use MAPseq to determine the projection patterns of over 3000 macaque BLA neurons.

Axonal Barcode Analysis of Pyramidal Tract Projections from Mouse Forelimb M1 and M2.

Forelimb-related areas of the motor cortex communicate directly to downstream areas in the brainstem and spinal cord via axons that project to and through the pyramidal tract (PT). To better understand the diversity of the brainstem branching patterns of these pyramidal tract projections, we used MAPseq, a molecular barcode technique for population-scale sampling with single-axon resolution. In experiments using mice of both sexes, we first confirmed prior results demonstrating the basic efficacy of axonal barcode identification of primary motor cortex (M1) PT-type axons, including corticobulbar (CBULB) and corticospinal (CSPI) subclasses.

High-throughput sequencing of single neuron projections reveals spatial organization in the olfactory cortex.

In most sensory modalities, neuronal connectivity reflects behaviorally relevant stimulus features, such as spatial location, orientation, and sound frequency. By contrast, the prevailing view in the olfactory cortex, based on the reconstruction of dozens of neurons, is that connectivity is random. Here, we used high-throughput sequencing-based neuroanatomical techniques to analyze the projections of 5,309 mouse olfactory bulb and 30,433 piriform cortex output neurons at single-cell resolution.

Cellular anatomy of the mouse primary motor cortex.

An essential step toward understanding brain function is to establish a structural framework with cellular resolution on which multi-scale datasets spanning molecules, cells, circuits and systems can be integrated and interpreted. Here, as part of the collaborative Brain Initiative Cell Census Network (BICCN), we derive a comprehensive cell type-based anatomical description of one exemplar brain structure, the mouse primary motor cortex, upper limb area (MOp-ul). Using genetic and viral labelling, barcoded anatomy resolved by sequencing, single-neuron reconstruction, whole-brain imaging and cloud-based neuroinformatics tools, we delineated the MOp-ul in 3D and refined its sublaminar organization.

Integrating barcoded neuroanatomy with spatial transcriptional profiling enables identification of gene correlates of projections.

Functional circuits consist of neurons with diverse axonal projections and gene expression. Understanding the molecular signature of projections requires high-throughput interrogation of both gene expression and projections to multiple targets in the same cells at cellular resolution, which is difficult to achieve using current technology. Here, we introduce BARseq2, a technique that simultaneously maps projections and detects multiplexed gene expression by in situ sequencing.

SYNPLA, a method to identify synapses displaying plasticity after learning.

Which neural circuits undergo synaptic changes when an animal learns? Although it is widely accepted that changes in synaptic strength underlie many forms of learning and memory, it remains challenging to connect changes in synaptic strength at specific neural pathways to specific behaviors and memories. Here we introduce SYNPLA (synaptic proximity ligation assay), a synapse-specific, high-throughput, and potentially brain-wide method capable of detecting circuit-specific learning-induced synaptic plasticity.

High-Throughput Mapping of Long-Range Neuronal Projection Using In Situ Sequencing.

Understanding neural circuits requires deciphering interactions among myriad cell types defined by spatial organization, connectivity, gene expression, and other properties. Resolving these cell types requires both single-neuron resolution and high throughput, a challenging combination with conventional methods. Here, we introduce barcoded anatomy resolved by sequencing (BARseq), a multiplexed method based on RNA barcoding for mapping projections of thousands of spatially resolved neurons in a single brain and relating those projections to other properties such as gene or Cre expression.

Sequencing the connectome.

Connectivity determines the function of neural circuits. Historically, circuit mapping has usually been viewed as a problem of microscopy, but no current method can achieve high-throughput mapping of entire circuits with single neuron precision. Here we describe a novel approach to determining connectivity.

A transgenic zebrafish liver tumor model with inducible Myc expression reveals conserved Myc signatures with mammalian liver tumors.

Myc is a pleiotropic transcription factor that is involved in many cellular activities relevant to carcinogenesis, including hepatocarcinogenesis. The zebrafish has been increasingly used to model human diseases and it is particularly valuable in helping to identify common and conserved molecular mechanisms in vertebrates. Here we generated a liver tumor model in transgenic zebrafish by liver-specific expression of mouse Myc using a Tet-On system.

Inducible and repressable oncogene-addicted hepatocellular carcinoma in Tet-on xmrk transgenic zebrafish.

Background & Aims: Liver cancer, mainly hepatocellular carcinoma, is a major malignancy and currently there are no effective treatment protocols due to insufficient understanding of hepatocarcinogenesis. As a potentially high-throughput and cost-effective experimental model, the zebrafish is increasingly recognized for disease studies. Here, we aim at using the zebrafish to generate a convenient hepatocellular carcinoma model.

A ΔRaf1-ER-inducible oncogenic zebrafish liver cell model identifies hepatocellular carcinoma signatures.

Although the underlying molecular mechanism of hepatocellular carcinoma remains unclear, signalling pathways essential in cell survival and growth are altered, including the Raf-MEK-MAPK pathway. This pathway can be activated by hepatitis B or C virus infections and the ectopic expression of the Raf-1 oncogene is frequently seen in hepatocellular carcinomas. In addition, the Raf-MEK-MAPK pathway was also shown to be deregulated in zebrafish liver tumours.

Comprehensive profiling of zebrafish hepatic proximal promoter CpG island methylation and its modification during chemical carcinogenesis.

Background: DNA methylation is an epigenetic mechanism associated with regulation of gene expression and it is modulated during chemical carcinogenesis. The zebrafish is increasingly employed as a human disease model; however there is a lack of information on DNA methylation in zebrafish and during fish tumorigenesis.

Results: A novel CpG island tiling array containing 44,000 probes, in combination with immunoprecipitation of methylated DNA, was used to achieve the first comprehensive methylation profiling of normal adult zebrafish liver.

Transgenic expression of walleye dermal sarcoma virus rv-cyclin (orfA) in zebrafish does not result in tissue proliferation.

Walleye dermal sarcoma (WDS) is a benign tumor of walleye fish that develops and completely regresses seasonally. The retrovirus associated with this disease, walleye dermal sarcoma virus, encodes three accessory genes, two of which, rv-cyclin (orfA) and orfb, are thought to play a role in tumor development. In this study, we attempted to recapitulate WDS development by expressing rv-cyclin in chimeric and stable transgenic zebrafish.

Transgenic expression of walleye dermal sarcoma virus rv-cyclin gene in zebrafish and its suppressive effect on liver tumor development after carcinogen treatment.

A retrovirus homologue gene of cellular cyclin D₁, walleye dermal sarcoma virus rv-cyclin gene (orf A or rv-cyclin), was expressed in the livers of zebrafish under the control of liver fatty acid-binding protein (lfabp) promoter. To prevent possible fatality caused by overexpression of the oncogene, the GAL4/upstream activation sequence (GAL4/UAS) system was used to maintain the transgenic lines. Thus, both GAL4-activator [Tg(lfabp:GAL4)] and UAS-effector [Tg(UAS:rvcyclin)] lines were generated, and the rv-cyclin gene was activated in the liver after crossing these two lines.

Delayed and restricted expression of UAS-regulated GFP gene in early transgenic zebrafish embryos by using the GAL4/UAS system.

A stable Tg(UAS:GFP) zebrafish line was generated and crossed with Tg(hsp70:GAL4) line, in which the GAL4 gene is under the control of an inducible zebrafish promoter derived from the heat shock 70 protein gene (hsp70). The dynamic green fluorescent protein (GFP) expression in early zebrafish embryos in the GAL4/UAS binary system was then investigated. We found that, at early developmental stages, expression of GFP effector gene was restricted and required a long recovery time to reach a detectable level.

Ornamental expression of red fluorescent protein in transgenic founders of white skirt tetra (Gymnocorymbus ternetzi).

Although the transgenic technology has been successfully used to generate fluorescent zebrafish and medaka for ornamental purposes, the practicability of the technology has not been demonstrated in other ornamental fish species. In the present study, we have tested the transgenic technology in a bona fide ornamental fish species, the white skirt tetra (Gymnocorymbus ternetzi). First, its embryonic development was briefly described.

Conservation of gene expression signatures between zebrafish and human liver tumors and tumor progression.

The zebrafish (Danio rerio) has been long advocated as a model for cancer research, but little is known about the real molecular similarities between zebrafish and human tumors. Comparative analysis of microarray data from zebrafish liver tumors with those from four human tumor types revealed molecular conservation at various levels between fish and human tumors. This approach provides a useful strategy for identifying an expression signature that is strongly associated with a disease phenotype.