Morphological diversity of single neurons in molecularly defined cell types.

Dendritic and axonal morphology reflects the input and output of neurons and is a defining feature of neuronal types1,2, yet our knowledge of its diversity remains limited. Here, to systematically examine complete single-neuron morphologies on a brain-wide scale, we established a pipeline encompassing sparse labelling, whole-brain imaging, reconstruction, registration and analysis. We fully reconstructed 1,741 neurons from cortex, claustrum, thalamus, striatum and other brain regions in mice. We identified 11 major projection neuron types with distinct morphological features and corresponding transcriptomic identities. Extensive projectional diversity was found within each of these major types, on the basis of which some types were clustered into more refined subtypes. This diversity follows a set of generalizable principles that govern long-range axonal projections at different levels, including molecular correspondence, divergent or convergent projection, axon termination pattern, regional specificity, topography, and individual cell variability. Although clear concordance with transcriptomic profiles is evident at the level of major projection type, fine-grained morphological diversity often does not readily correlate with transcriptomic subtypes derived from unsupervised clustering, highlighting the need for single-cell cross-modality studies. Overall, our study demonstrates the crucial need for quantitative description of complete single-cell anatomy in cell-type classification, as single-cell morphological diversity reveals a plethora of ways in which different cell types and their individual members may contribute to the configuration and function of their respective circuits.

Runx1 is sufficient but not required for cardiomyocyte cell-cycle activation.

Factors responsible for cardiomyocyte proliferation could serve as potential therapeutics to stimulate endogenous myocardial regeneration following insult, such as ischemic injury. A previously published forward genetics approach on cardiomyocyte cell cycle and ploidy led us to the transcription factor, Runx1. Here, we examine the effect of Runx1 on cardiomyocyte cell cycle during postnatal development and cardiac regeneration using cardiomyocyte-specific gain- and loss-of-function mouse models. RUNX1 is expressed in cardiomyocytes during early postnatal life, decreases to negligible levels by 3 wk of age, and increases upon myocardial injury, all consistent with observed rates of cardiomyocyte cell-cycle activity. Loss of Runx1 transiently stymied cardiomyocyte cell-cycle activity during normal postnatal development, a result that corrected itself and did not extend to the context of neonatal heart regeneration. On the other hand, cardiomyocyte-specific Runx1 overexpression resulted in an expansion of diploid cardiomyocytes in uninjured hearts and expansion of 4 N cardiomyocytes in the context of neonatal cardiac injury, suggesting Runx1 overexpression is sufficient to induce cardiomyocyte cell-cycle responses. Persistent overexpression of Runx1 for >1 mo continued to promote cardiomyocyte cell-cycle activity resulting in substantial hyperpolyploidization (≥8 N DNA content). This persistent cell-cycle activation was accompanied by ventricular dilation and adverse remodeling, raising the concern that continued cardiomyocyte cell cycling can have detrimental effects.NEW & NOTEWORTHY Runx1 is sufficient but not required for cardiomyocyte cell cycle.

Mesenchymal Stromal Cells Facilitate Tip Cell Fusion Downstream of BMP-Mediated Venous Angiogenesis-Brief Report.

BACKGROUND: The goal of this study was to identify and characterize cell-cell interactions that facilitate endothelial tip cell fusion downstream of BMP (bone morphogenic protein)-mediated venous plexus formation. METHODS: High resolution and time-lapse imaging of transgenic reporter lines and loss-of-function studies were carried out to study the involvement of mesenchymal stromal cells during venous angiogenesis. RESULTS: BMP-responsive stromal cells facilitate timely and precise fusion of venous tip cells during developmental angiogenesis. CONCLUSIONS: Stromal cells are required for anastomosis of venous tip cells in the embryonic caudal hematopoietic tissue.

Transdifferentiation of fast skeletal muscle into functional endothelium in vivo by transcription factor Etv2.

Etsrp/Etv2 (Etv2) is an evolutionarily conserved master regulator of vascular development in vertebrates. Etv2 deficiency prevents the proper specification of the endothelial cell lineage, while its overexpression causes expansion of the endothelial cell lineage in the early embryo or in embryonic stem cells. We hypothesized that Etv2 alone is capable of transdifferentiating later somatic cells into endothelial cells. Using heat shock inducible Etv2 transgenic zebrafish, we demonstrate that Etv2 expression alone is sufficient to transdifferentiate fast skeletal muscle cells into functional blood vessels. Following heat treatment, fast skeletal muscle cells turn on vascular genes and repress muscle genes. Time-lapse imaging clearly shows that muscle cells turn on vascular gene expression, undergo dramatic morphological changes, and integrate into the existing vascular network. Lineage tracing and immunostaining confirm that fast skeletal muscle cells are the source of these newly generated vessels. Microangiography and observed blood flow demonstrated that this new vasculature is capable of supporting circulation. Using pharmacological, transgenic, and morpholino approaches, we further establish that the canonical Wnt pathway is important for induction of the transdifferentiation process, whereas the VEGF pathway provides a maturation signal for the endothelial fate. Additionally, overexpression of Etv2 in mammalian myoblast cells, but not in other cell types examined, induced expression of vascular genes. We have demonstrated in zebrafish that expression of Etv2 alone is sufficient to transdifferentiate fast skeletal muscle into functional endothelial cells in vivo. Given the evolutionarily conserved function of this transcription factor and the responsiveness of mammalian myoblasts to Etv2, it is likely that mammalian muscle cells will respond similarly.

Etsrp/Etv2 is directly regulated by Foxc1a/b in the zebrafish angioblast.

RATIONALE: Endothelial cells are developmentally derived from angioblasts specified in the mesodermal germ cell layer. The transcription factor etsrp/etv2 is at the top of the known genetic hierarchy for angioblast development. The transcriptional events that induce etsrp expression and angioblast specification are not well understood. OBJECTIVE: We generated etsrp:gfp transgenic zebrafish and used them to identify regulatory regions and transcription factors critical for etsrp expression and angioblast specification from mesoderm. METHODS AND RESULTS: To investigate the mechanisms that initiate angioblast cell transcription during embryogenesis, we have performed promoter analysis of the etsrp locus in zebrafish. We describe three enhancer elements sufficient for endothelial gene expression when place in front of a heterologous promoter. The deletion of all 3 regulatory regions led to a near complete loss of endothelial expression from the etsrp promoter. One of the enhancers, located 2.3 kb upstream of etsrp contains a consensus FOX binding site that binds Foxc1a and Foxc1b in vitro by EMSA and in vivo using ChIP. Combined knockdown of foxc1a/b, using morpholinos, led to a significant decrease in etsrp expression at early developmental stages as measured by quantitative reverse transcriptase-polymerase chain reaction and in situ hybridization. Decreased expression of primitive erythrocyte genes scl and gata1 was also observed, whereas pronephric gene pax2a was relatively normal in expression level and pattern. CONCLUSIONS: These findings identify mesodermal foxc1a/b as a direct upstream regulator of etsrp in angioblasts. This establishes a new molecular link in the process of mesoderm specification into angioblast.

LCM-Seq for Retinal Cell Layer-Specific Responses During Optic Nerve Regeneration.

LCM-seq is a powerful tool for gene expression analysis from individual or groups of cells that can be spatially isolated. Within the visual system, retinal ganglion cells (RGCs), the cells that connect the eye to the brain through the optic nerve, reside in the retinal ganglion cell layer of the retina. This well-defined location provides a unique opportunity to harvest RNA by laser capture microdissection (LCM) from a highly enriched cell population. Using this method, it is possible to explore transcriptome-wide changes in gene expression following optic nerve injury. In the zebrafish model, this method can be used to identify molecular events driving successful optic nerve regeneration in contrast to mammals that fail to regenerate axons in the central nervous system. Here we provide a method for LCM from the different retinal layers of zebrafish following optic nerve injury and during the process of optic nerve regeneration. Purified RNA from this protocol is sufficient for RNA-seq or other downstream analysis.

Intraocular Axon Regeneration in a Model of Penetrating Eye Injury.

Purpose: Penetrating eye injuries commonly cause permanent loss of vision in patients. Unlike mammals, zebrafish can regenerate both damaged tissue and severed axons in the central nervous system. Here, we present a tractable adult zebrafish model to study intraocular axon regeneration after penetrating eye injury. Methods: To create consistent penetrating intraocular injuries, pins of standardized diameters were inserted into the eye through the cornea and penetrating the retina but not the underlying sclera. Transgenic gap43:GFP reporter fish were used to preferentially label retinal ganglion cells (RGCs) that respond to injury with regenerating axons. Retinas were fixed and flat mounted at various times postinjury to examine injury size, number of green fluorescent protein (GFP)-positive cells and axons, axonal varicosities, and rate of regeneration to the optic nerve head. Intraocular injection of colchicine was used to inhibit axon outgrow as a proof of principle that this method can be used to screen effects of pharmacological agents on intraocular axon regeneration. Results: Penetrating injury to the zebrafish retina results in robust axon regeneration by RGCs around and beyond the site of injury. The gap43:GFP transgene allows visualization of individual or small bundles of axons with varicosities and growth cones easily observable. Regeneration proceeded with most, if not all, axons reaching the optic nerve head by 3-day postinjury. A single intraocular injection of colchicine a day after injury was sufficient to delay axon regeneration at 2-days postinjury. Surprisingly, we identified a stereotypically located population of circumferential projecting neurons within the retina that upregulate gap43:GFP expression after injury. Conclusions: Penetrating injury to the adult gap43:GFP transgenic zebrafish eye is a model of successful intraocular axon regeneration. The pharmacological and genetic tools available for this organism should make it a powerful tool for dissecting the cellular, molecular, and genetic mechanisms of axon regeneration in the intraocular environment.

Metabolomics dataset of zebrafish optic nerve regeneration after injury.

Zebrafish (Danio rerio) have the capacity for successful adult optic nerve regeneration. In contrast, mammals lack this intrinsic ability and undergo irreversible neurodegeneration seen in glaucoma and other optic neuropathies. Optic nerve regeneration is often studied using optic nerve crush, a mechanical neurodegenerative model. Untargeted metabolomic studies within successful regenerative models are deficient. Evaluation of tissue metabolomic changes in active zebrafish optic nerve regeneration can elucidate prioritized metabolite pathways that can be targeted in mammalian systems for therapeutic development. Female and male (6 month to 1 year old wild type) right zebrafish optic nerves were crushed and collected three days after. Contralateral, uninjured optic nerves were collected as controls. The tissue was dissected from euthanized fish and frozen on dry ice. Samples were pooled for each category (female crush, female control, male crush, male control) and pooled at n = 31 to obtain sufficient metabolite concentrations for analysis. Optic nerve regeneration at 3 days post crush was demonstrated by microscope visualization of GFP fluorescence in Tg(gap43:GFP) transgenic fish. Metabolites were extracted using a Precellys Homogenizer and a serial extraction method: (1) 1:1 Methanol/Water and (2) 8:1:1 Acetonitrile/Methanol/Acetone. Metabolites were analyzed by untargeted liquid chromatography-mass spectrometry (LC MS-MS) profiling using a Q-Exactive Orbitrap instrument coupled with Vanquish Horizon Binary UHPLC LC-MS system. Metabolites were identified and quantified using Compound Discoverer 3.3 and isotopic internal metabolites standards.

Tuba1a gene expression is regulated by KLF6/7 and is necessary for CNS development and regeneration in zebrafish.

We report that knockdown of the alpha1 tubulin isoform Tuba1a, but not the highly related Tuba1b, dramatically impedes nervous system formation during development and RGC axon regeneration following optic nerve injury in adults. Within the tuba1a promoter, a G/C-rich element was identified that is necessary for tuba1a induction during RGC differentiation and optic axon regeneration. KLF6a and 7a, which we previously reported are essential for optic axon regeneration (Veldman et al., 2007), bind this G/C-rich element and transactivate the tuba1a promoter. In vivo knockdown of KLF6a and 7a attenuate regeneration-dependent activation of the endogenous tuba1a and p27 genes. These results suggest tuba1a expression is necessary for CNS development and regeneration and that KLF6a and 7a mediate their effects, at least in part, via transcriptional control of tuba1a promoter activity.

Lipidomics dataset of Danio rerio optic nerve regeneration model.

The right optic nerve of adult, 6 month to 1 year old, female and male Danio rerio were crushed and collected three days after. Matching controls of uninjured left optic nerves were also collected. The tissue was dissected from euthanized fish and frozen on dry ice. Samples were pooled for each category (female crush, female control, male crush, male control) n = 24 to obtain sufficient tissue for analysis. The brain from one male fish was also collected for control/calibration. Lipid extraction was done with the Bligh and Dyer [1] method, followed by untargeted liquid chromatography-mass spectrometry (LC MS-MS) lipid profiling using a Q-Exactive Orbitrap instrument coupled with Vanquish Horizon Binary UHPLC LC-MS system. The lipids were identified and quantified with LipidSearch 4.2.21 and the statistical analysis was conducted through Metaboanalyst 5.0. This data is available at Metabolomics Workbench, Study ID ST001725.

Brainwide Genetic Sparse Cell Labeling to Illuminate the Morphology of Neurons and Glia with Cre-Dependent MORF Mice.

Cajal recognized that the elaborate shape of neurons is fundamental to their function in the brain. However, there are no simple and generalizable genetic methods to study neuronal or glial cell morphology in the mammalian brain. Here, we describe four mouse lines conferring Cre-dependent sparse cell labeling based on mononucleotide repeat frameshift (MORF) as a stochastic translational switch. Notably, the optimized MORF3 mice, with a membrane-bound multivalent immunoreporter, confer Cre-dependent sparse and bright labeling of thousands of neurons, astrocytes, or microglia in each brain, revealing their intricate morphologies. MORF3 mice are compatible with imaging in tissue-cleared thick brain sections and with immuno-EM. An analysis of 151 MORF3-labeled developing retinal horizontal cells reveals novel morphological cell clusters and axonal maturation patterns. Our study demonstrates a conceptually novel, simple, generalizable, and scalable mouse genetic solution to sparsely label and illuminate the morphology of genetically defined neurons and glia in the mammalian brain.

Highly-restricted, cell-specific expression of the simian CMV-IE promoter in transgenic zebrafish with age and after heat shock.

Promoters with high levels of ubiquitous expression are of significant utility in the production of transgenic animals and cell lines. One such promoter is derived from the human cytomegalovirus immediate early (CMV-IE) gene. We sought to ascertain if the simian CMV-IE promoter (sCMV), used extensively in non-mammalian vertebrate research, also directs intense, widespread expression when stably introduced into zebrafish. Analysis of sCMV-driven expression revealed a temporal and spatial pattern not predicted by studies using the hCMV promoter in other transgenic animals or by observations of early F0 embryos expressing injected sCMV-reporter plasmids. Unexpectedly, in transgenic fish produced by both integration of linearized plasmid or Tol2-mediated transgenesis, sCMV promoter expression was generally observed in a small population of cells in telencephalon and spinal cord between days 2 and 7, and was thereafter confined to discrete regions of CNS that included the olfactory bulb, retina, cerebellum, spinal cord, and lateral line. In skeletal muscle, intense transgene expression was not observed until well into adulthood (>2-3 months post-fertilization). One final unexpected characteristic of the sCMV promoter in stable transgenic fish was tissue-specific responsiveness of the promoter to heat shock at both embryonic and adult stages. These data suggest that, in the context of stable transgenesis, the simian CMV-IE gene promoter responds differently to intracellular regulatory forces than other characterized CMV promoters.

Precise segmentation of densely interweaving neuron clusters using G-Cut.

Characterizing the precise three-dimensional morphology and anatomical context of neurons is crucial for neuronal cell type classification and circuitry mapping. Recent advances in tissue clearing techniques and microscopy make it possible to obtain image stacks of intact, interweaving neuron clusters in brain tissues. As most current 3D neuronal morphology reconstruction methods are only applicable to single neurons, it remains challenging to reconstruct these clusters digitally. To advance the state of the art beyond these challenges, we propose a fast and robust method named G-Cut that is able to automatically segment individual neurons from an interweaving neuron cluster. Across various densely interconnected neuron clusters, G-Cut achieves significantly higher accuracies than other state-of-the-art algorithms. G-Cut is intended as a robust component in a high throughput informatics pipeline for large-scale brain mapping projects.

Gene expression analysis of zebrafish retinal ganglion cells during optic nerve regeneration identifies KLF6a and KLF7a as important regulators of axon regeneration.

Unlike mammals, teleost fish are able to mount an efficient and robust regenerative response following optic nerve injury. Although it is clear that changes in gene expression accompany axonal regeneration, the extent of this genomic response is not known. To identify genes involved in successful nerve regeneration, we analyzed gene expression in zebrafish retinal ganglion cells (RGCs) regenerating their axons following optic nerve injury. Microarray analysis of RNA isolated by laser capture microdissection from uninjured and 3-day post-optic nerve injured RGCs identified 347 up-regulated and 29 down-regulated genes. Quantitative RT-PCR and in situ hybridization were used to verify the change in expression of 19 genes in this set. Gene ontological analysis of the data set suggests regenerating neurons up-regulate genes associated with RGC development. However, not all regeneration-associated genes are expressed in differentiating RGCs indicating the regeneration is not simply a recapitulation of development. Knockdown of six highly induced regeneration-associated genes identified two, KLF6a and KLF7a, that together were necessary for robust RGC axon re-growth. These results implicate KLF6a and KLF7a as important mediators of optic nerve regeneration and suggest that not all induced genes are essential to mount a regenerative response.

Zebrafish as a developmental model organism for pediatric research.

Zebrafish has many advantages as a model of human pediatric research. Given the physical and ethical problems with performing experiments on human patients, biomedical research has focused on using model organisms to study biologic processes conserved between humans and lower vertebrates. The most common model organisms are small mammals, usually rats and mice. Although these models have significant advantages, they are also expensive to maintain, difficult to manipulate embryonically, and limited for large-scale genetic studies. The zebrafish model nicely complements these deficiencies in mammalian experimental models. The low cost, small size, and external development of zebrafish make it an excellent model for vertebrate development biology. Techniques for large-scale genome mutagenesis and gene mapping, transgenesis, protein overexpression or knockdown, cell transplantation and chimeric embryo analysis, and chemical screens have immeasurably increased the power of this model organism. It is now possible to rapidly determine the developmental function of a gene of interest in vivo, and then identify genetic and chemical modifiers of the processes involved. Discoveries made in zebrafish can be further validated in mammals. With novel technologies being regularly developed, the zebrafish is poised to significantly improve our understanding of vertebrate development under normal and pathologic conditions.

Molecular insights into cortico-striatal miscommunications in Huntington's disease.

Huntington's disease (HD), a dominantly inherited neurodegenerative disease, is defined by its genetic cause, a CAG-repeat expansion in the HTT gene, its motor and psychiatric symptomology and primary loss of striatal medium spiny neurons (MSNs). However, the molecular mechanisms from genetic lesion to disease phenotype remain largely unclear. Mouse models of HD have been created that exhibit phenotypes partially recapitulating those in the patient, and specifically, cortico-striatal disconnectivity appears to be a shared pathogenic event shared by HD mouse models and patients. Molecular studies have begun to unveil converging molecular and cellular pathogenic mechanisms that may account for cortico-striatal miscommunication in various HD mouse models. Systems biological approaches help to illuminate synaptic molecular networks as a nexus for HD cortio-striatal pathogenesis, and may offer new candidate targets to modify the disease.

Huntington's Disease: Nuclear Gatekeepers Under Attack.

In this issue of Neuron, Gasset-Rosa et al. (2017) and Grima et al. (2017) describe defects in the nuclear pore complex and impaired nucleocytoplasmic transport in Huntington's disease (HD). The findings suggest that erosion of nuclear gatekeeping function, which is found in normal brain aging, may play an important role in the pathogenesis of multiple neurodegenerative disorders, including HD.

ETV2 expression increases the efficiency of primitive endothelial cell derivation from human embryonic stem cells.

BACKGROUND: Endothelial cells line the luminal surface of blood vessels and form a barrier between the blood and other tissues of the body. Ets variant 2 (ETV2) is transiently expressed in both zebrafish and mice and is necessary and sufficient for vascular endothelial cell specification. Overexpression of this gene in early zebrafish and mouse embryos results in ectopic appearance of endothelial cells. Ectopic expression of ETV2 in later development results in only a subset of cells responding to the signal. FINDINGS: We have examined the expression pattern of ETV2 in differentiating human embryonic stem cells (ESCs) to determine when the peak of ETV2 expression occurs. We show that overexpression of ETV2 in differentiating human ESC is able to increase the number of endothelial cells generated when administered during or after the endogenous peak of gene expression. CONCLUSIONS: Addition of exogenous ETV2 to human ESCs significantly increased the number of cells expressing angioblast genes without arterial or venous specification. This may be a viable solution to generate in vitro endothelial cells for use in research and in the clinic.

The N17 domain mitigates nuclear toxicity in a novel zebrafish Huntington's disease model.

BACKGROUND: Although the genetic cause for Huntington's disease (HD) has been known for over 20 years, the mechanisms that cause the neurotoxicity and behavioral symptoms of this disease are not well understood. One hypothesis is that N-terminal fragments of the HTT protein are the causative agents in HD and that peptide sequences adjacent to the poly-glutamine (Q) repeats modify its toxicity. Here we test the function of the N-terminal 17 amino acids (N17) in the context of the exon 1 fragment of HTT in a novel, inducible zebrafish model of HD. RESULTS: Deletion of N17 coupled with 97Q expansion (mHTT-ΔN17-exon1) resulted in a robust, rapidly progressing movement deficit, while fish with intact N17 and 97Q expansion (mHTT-exon1) have more delayed-onset movement deficits with slower progression. The level of mHTT-ΔN17-exon1 protein was significantly higher than mHTT-exon1, although the mRNA level of each transgene was marginally different, suggesting that N17 may regulate HTT protein stability in vivo. In addition, cell lineage specific induction of the mHTT-ΔN17-exon1 transgene in neurons was sufficient to recapitulate the consequences of ubiquitous transgene expression. Within neurons, accelerated nuclear accumulation of the toxic HTT fragment was observed in mHTT-ΔN17-exon1 fish, demonstrating that N17 also plays an important role in sub-cellular localization in vivo. CONCLUSIONS: We have developed a novel, inducible zebrafish model of HD. These animals exhibit a progressive movement deficit reminiscent of that seen in other animal models and human patients. Deletion of the N17 terminal amino acids of the huntingtin fragment results in an accelerated HD-like phenotype that may be due to enhanced protein stability and nuclear accumulation of HTT. These transgenic lines will provide a valuable new tool to study mechanisms of HD at the behavioral, cellular, and molecular levels. Future experiments will be focused on identifying genetic modifiers, mechanisms and therapeutics that alleviate polyQ aggregation in the nucleus of neurons.