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1.  Canonical WNT signaling components in vascular development and barrier formation 
The Journal of Clinical Investigation  2014;124(9):3825-3846.
Canonical WNT signaling is required for proper vascularization of the CNS during embryonic development. Here, we used mice with targeted mutations in genes encoding canonical WNT pathway members to evaluate the exact contribution of these components in CNS vascular development and in specification of the blood-brain barrier (BBB) and blood-retina barrier (BRB). We determined that vasculature in various CNS regions is differentially sensitive to perturbations in canonical WNT signaling. The closely related WNT signaling coreceptors LDL receptor–related protein 5 (LRP5) and LRP6 had redundant functions in brain vascular development and barrier maintenance; however, loss of LRP5 alone dramatically altered development of the retinal vasculature. The BBB in the cerebellum and pons/interpeduncular nuclei was highly sensitive to decrements in canonical WNT signaling, and WNT signaling was required to maintain plasticity of barrier properties in mature CNS vasculature. Brain and retinal vascular defects resulting from ablation of Norrin/Frizzled4 signaling were ameliorated by stabilizing β-catenin, while inhibition of β-catenin–dependent transcription recapitulated the vascular development and barrier defects associated with loss of receptor, coreceptor, or ligand, indicating that Norrin/Frizzled4 signaling acts predominantly through β-catenin–dependent transcriptional regulation. Together, these data strongly support a model in which identical or nearly identical canonical WNT signaling mechanisms mediate neural tube and retinal vascularization and maintain the BBB and BRB.
doi:10.1172/JCI76431
PMCID: PMC4151216  PMID: 25083995
2.  Cellular resolution maps of X-chromosome inactivation: implications for neural development, function, and disease 
Neuron  2014;81(1):103-119.
Female eutherian mammals use X-chromosome inactivation (XCI) to epigenetically regulate gene expression from ~4% of genes. To quantitatively map the topography of XCI for defined cell types at single cell resolution, we have generated female mice that carry X-linked, Cre-activated, and nuclear-localized fluorescent reporters – GFP on one X-chromosome and tdTomato on the other. Using these reporters in combination with different Cre drivers we have defined the topographies of XCI mosaicism for multiple CNS cell types and of retinal vascular dysfunction in a model of Norrie Disease. Depending on cell type, fluctuations in the XCI mosaic are observed over a wide range of spatial scales, from neighboring cells to left vs. right sides of the body. These data imply a major role for XCI in generating female-specific, genetically directed, stochastic diversity in eutherian mammals on spatial scales that would be predicted to affect CNS function within and between individuals.
doi:10.1016/j.neuron.2013.10.051
PMCID: PMC3950970  PMID: 24411735
3.  Complete morphologies of basal forebrain cholinergic neurons in the mouse 
eLife  2014;3:e02444.
The basal forebrain cholinergic system modulates neuronal excitability and vascular tone throughout the cerebral cortex and hippocampus. This system is severely affected in Alzheimer's disease (AD), and drug treatment to enhance cholinergic signaling is widely used as symptomatic therapy in AD. Defining the full morphologies of individual basal forebrain cholinergic neurons has, until now, been technically beyond reach due to their large axon arbor sizes. Using genetically-directed sparse labeling, we have characterized the complete morphologies of basal forebrain cholinergic neurons in the mouse. Individual arbors were observed to span multiple cortical columns, and to have >1000 branch points and total axon lengths up to 50 cm. In an AD model, cholinergic axons were slowly lost and there was an accumulation of axon-derived material in discrete puncta. Calculations based on published morphometric data indicate that basal forebrain cholinergic neurons in humans have a mean axon length of ∼100 meters.
DOI: http://dx.doi.org/10.7554/eLife.02444.001
eLife digest
The human brain is made up of roughly 80 to 100 billion neurons, organized into extensive networks. Each neuron consists of a number of components: a cell body, which contains the nucleus; numerous short protrusions from the cell body called dendrites; and a long thin structure called an axon that carries the electrical signals generated in the cell body and the dendrites to the next neuron in the network.
One of the most studied networks in the human brain is the basal forebrain network, which is made up of large neurons that communicate with one another using a chemical transmitter called acetylcholine. This network has a key role in cognition, and its neurons are among the first to degenerate in Alzheimer's disease. However, relatively little is known about the structure of these ‘cholinergic’ neurons because their large size makes them difficult to study using standard techniques.
Now, Wu et al. have visualized, for the first time, the complete 3D structure of cholinergic neurons in the mouse forebrain. The mice in question had been genetically modified so that only ten or so of their many thousands of cholinergic neurons expressed a distinctive ‘marker’ protein. This made it possible to distinguish these neurons from surrounding brain tissue in order to visualize their structures. The resulting pictures clearly illustrate the neurons' complexity, with individual axons in adult mice displaying up to 1000 branches.
Measurements showed that each cholinergic axon in the mouse brain is roughly 30 centimeters long, even though the brain itself is less than 2 centimeters from front to back. Based on measurements by other researchers, Wu et al. calculated that the axons of single cholinergic neurons in the human brain are about 100 meters long on average.
The extreme length and complex branching structure of cholinergic forebrain neurons helps to explain why each neuron is able to modulate the activity of many others in the network. It could also explain their vulnerability to degeneration, as the need to transport materials over such long distances may limit the ability of these neurons to respond to damage.
DOI: http://dx.doi.org/10.7554/eLife.02444.002
doi:10.7554/eLife.02444
PMCID: PMC4038840  PMID: 24894464
Cre; LoxP; Alzheimer's disease; neurodegeneration; cerebral cortex; axon arbor; mouse
4.  Signaling by Sensory Receptors 
Sensory systems detect small molecules, mechanical perturbations, or radiation via the activation of receptor proteins and downstream signaling cascades in specialized sensory cells. In vertebrates, the two principal categories of sensory receptors are ion channels, which mediate mechanosensation, thermosensation, and acid and salt taste; and G-protein-coupled receptors (GPCRs), which mediate vision, olfaction, and sweet, bitter, and umami tastes. GPCR-based signaling in rods and cones illustrates the fundamental principles of rapid activation and inactivation, signal amplification, and gain control. Channel-based sensory systems illustrate the integration of diverse modulatory signals at the receptor, as seen in the thermosensory/pain system, and the rapid response kinetics that are possible with direct mechanical gating of a channel. Comparisons of sensory receptor gene sequences reveal numerous examples in which gene duplication and sequence divergence have created novel sensory specificities. This is the evolutionary basis for the observed diversity in temperature- and ligand-dependent gating among thermosensory channels, spectral tuning among visual pigments, and odorant binding among olfactory receptors. The coding of complex external stimuli by a limited number of sensory receptor types has led to the evolution of modality-specific and species-specific patterns of retention or loss of sensory information, a filtering operation that selectively emphasizes features in the stimulus that enhance survival in a particular ecological niche. The many specialized anatomic structures, such as the eye and ear, that house primary sensory neurons further enhance the detection of relevant stimuli.
In vertebrates, G-protein-coupled receptors predominate as stimulus detectors in visual and olfactory receptor cells, whereas ion channels mainly detect auditory and somatosensory stimuli.
doi:10.1101/cshperspect.a005991
PMCID: PMC3249628  PMID: 22110046
5.  Frizzled3 controls axonal development in distinct populations of cranial and spinal motor neurons 
eLife  2013;2:e01482.
Disruption of the Frizzled3 (Fz3) gene leads to defects in axonal growth in the VIIth and XIIth cranial motor nerves, the phrenic nerve, and the dorsal motor nerve in fore- and hindlimbs. In Fz3−/− limbs, dorsal axons stall at a precise location in the nerve plexus, and, in contrast to the phenotypes of several other axon path-finding mutants, Fz3−/− dorsal axons do not reroute to other trajectories. Affected motor neurons undergo cell death 2 days prior to the normal wave of developmental cell death that coincides with innervation of muscle targets, providing in vivo evidence for the idea that developing neurons with long-range axons are programmed to die unless their axons arrive at intermediate targets on schedule. These experiments implicate planar cell polarity (PCP) signaling in motor axon growth and they highlight the question of how PCP proteins, which form cell–cell complexes in epithelia, function in the dynamic context of axonal growth.
DOI: http://dx.doi.org/10.7554/eLife.01482.001
eLife digest
For the nervous system to become wired up correctly, neurons within the developing embryo must project over long distances to form connections with remote targets. They do this by lengthening their axons—the ‘cables’ along which electrical signals flow—and some axons in adult humans can grow to be more than 1 metre long.
This type of long-range pathfinding activity is particularly common for neurons that control movement, as many of these neurons must establish connections with muscles that are some distance away from the brain. For example, motor neurons in the brainstem form connections with muscles in the face to control facial expressions, while motor neurons in parts of the spinal cord project to muscles in the limbs. Multiple signaling pathways tell the developing axons which direction to grow en route to their final targets.
Now, Hua et al. have shown that an evolutionarily conserved protein called Frizzled3 is also involved in this process. In mouse embryos that lacked Frizzled3, the motor nerves that control breathing and limb movements were thinner than those in normal mice. In the mutant animals, many motor axons failed to form connections with their targets. Instead, these axons came to an abrupt halt midway along their intended paths and the neurons from which they originated died soon afterwards. These experiments support the idea that developing neurons are programmed to die unless their axons progress on the appropriate schedule.
As well as increasing our knowledge of the networks of connections that form within the developing mammalian nervous system, the work of Hua et al. provides new insights into some of the molecular mechanisms by which these connections are established.
DOI: http://dx.doi.org/10.7554/eLife.01482.002
doi:10.7554/eLife.01482
PMCID: PMC3865743  PMID: 24347548
planar cell polarity (PCP); axon growth; limb innervation; neural crest; cell death; muscle atrophy; Mouse
6.  Norrin/Frizzled4 signaling in retinal vascular development and blood brain barrier plasticity 
Cell  2012;151(6):1332-1344.
Norrin/Frizzled4 (Fz4) signaling activates the canonical Wnt pathway to control retinal vascular development. Using genetically engineered mice, we show that precocious Norrin production leads to premature retinal vascular invasion and delayed Norrin production leads to characteristic defects in intra-retinal vascular architecture. In genetic mosaics, wild type endothelial cells (ECs) instruct neighboring Fz4−/− ECs to produce an architecturally normal mosaic vasculature, a cell non-autonomous effect. However, over the ensuing weeks, Fz4−/− ECs are selectively eliminated from the mosaic vasculature, implying the existence of a quality control program that targets defective ECs. In the adult retina and cerebellum, gain or loss of Norrin/Fz4 signaling results in a cell-autonomous gain or loss, respectively, of blood retina barrier (BRB) and blood brain barrier (BBB) function, indicating an ongoing requirement for Frizzled signaling in barrier maintenance and substantial plasticity in mature CNS vascular structure.
doi:10.1016/j.cell.2012.10.042
PMCID: PMC3535266  PMID: 23217714
7.  How to draw the line in biomedical research 
eLife  2013;2:e00638.
The use of the least squares method to calculate the best-fitting line through a two-dimensional scatter plot typically requires the user to assume that one of the variables depends on the other. However, in many cases the relationship between the two variables is more complex, and it is not valid to say that one variable is independent and the other is dependent. When analysing such data researchers should consider plotting the three regression lines that can be calculated for any two-dimensional scatter plot.
doi:10.7554/eLife.00638
PMCID: PMC3601633  PMID: 23538852
Tutorial; statistics; publishing
8.  Morphologic diversity of cutaneous sensory afferents revealed by genetically directed sparse labeling 
eLife  2012;1:e00181.
The diversity of cutaneous sensory afferents has been studied by many investigators using behavioral, physiologic, molecular, and genetic approaches. Largely missing, thus far, is an analysis of the complete morphologies of individual afferent arbors. Here we present a survey of cutaneous sensory arbor morphologies in hairy skin of the mouse using genetically-directed sparse labeling with a sensory neuron-specific alkaline phosphatase reporter. Quantitative analyses of 719 arbors, among which 77 were fully reconstructed, reveal 10 morphologically distinct types. Among the two types with the largest arbors, one contacts ∼200 hair follicles with circumferential endings and a second is characterized by a densely ramifying arbor with one to several thousand branches and a total axon length between one-half and one meter. These observations constrain models of receptive field size and structure among cutaneous sensory neurons, and they raise intriguing questions regarding the cellular and developmental mechanisms responsible for this morphological diversity.
DOI: http://dx.doi.org/10.7554/eLife.00181.001
eLife digest
Sensory neurons carry information from sensory cells in the eyes, ears and other sensory organs to the brain and spinal cord so that they can coordinate the body's response to its environment and various stimuli. The sensory organs responsible for four of the traditional senses—vision, hearing, smell and taste—are relatively small and self-contained: however, the sensory organ responsible for touch is as big as the body itself. Moreover, a variety of many different types of sensory cells in the skin allow the body to respond to temperature, pain, itches and a range of other external stimuli.
Despite more than a century of research, relatively little is known about the morphology of the complex networks (arbors) of sensory neurons that send signals towards the central nervous system. This is mainly due to difficulties involved in imaging intact skin, the way that different arbors overlap and intermingle, and the relatively large distances that separate the bodies of neuronal cells and the farthest reaches of their arbors.
Wu et al. employed an imaging method that exploits the Cre-Lox system that is already widely used in genetics. In this approach a Cre enzyme is used to remove a region of DNA that is flanked by two genetically engineered Lox sequences. Wu et al. used a gene that codes for an enzyme marker (alkaline phosphatase) that previous investigators had into the DNA of mice. The gene was inserted in such a way that it was only expressed in sensory neurons that innervate the skin when Cre-Lox recombination had removed an adjacent segment of DNA. Moreover, Wu et al. used this reporter gene in combination with a modified Cre enzyme that only enters the nuclei of cells in the presence of a drug (Tamoxifen), so the probability that the marker gene is expressed is determined by the concentration of Tamoxifen. By administering a low level of Tamoxifen to pregnant mice, it was possible to label a very small number of sensory neurons in each embryo. Individual neurons that express the alkaline phosphatase marker were visualized with a histochemical reaction that rendered them dark purple. The remainder of the tissue remained unstained.
Based on quantitative analyses of the morphologies of more than 700 arbors, Wu et al. identified 10 distinct types of neurons. Of the two types of neurons with the largest arbors, one makes contact with ∼200 hair follicles, with the nerve endings completely encircling the follicles; the other type of arbor contains several thousand branches, with a total length for all of the branches summing to as much as one meter in length. The next challenge is to study the morphologies of neurons in tissues other than the skin, and also the neurons involved in other sensory systems, and to explore the cellular and developmental mechanisms responsible for the morphological diversity found in these initial experiments.
DOI: http://dx.doi.org/10.7554/eLife.00181.002
doi:10.7554/eLife.00181
PMCID: PMC3524796  PMID: 23256042
skin; neuronal morphology; sparse labeling; receptive field; Brn3a; Mouse
9.  Combinatorial expression of Brn3 transcription factors in somatosensory neurons: genetic and morphologic analysis 
The three members of the Brn3 family of POU-domain transcription factors (Brn3a/Pou4f1, Brn3b/Pou4f2, and Brn3c/Pou4f3) are expressed in overlapping subsets of visual, auditory/vestibular, and somatosensory neurons. Using unmarked Brn3 null alleles and Brn3 conditional alleles in which gene loss is coupled to expression of an alkaline phosphatase reporter, together with sparse Cre-mediated recombination, we describe (1) the overlapping patterns of Brn3 gene expression in somatosensory neurons, (2) the manner in which these patterns correlate with molecular markers, peripheral afferent arbor morphologies, and dorsal horn projections, and (3) the consequences for these neurons of deleting individual Brn3 genes in the mouse. We observe broad expression of Brn3a among DRG neurons, but subtype-restricted expression of Brn3b and Brn3c. We also observe a nearly complete loss of hair follicle-associated sensory endings among Brn3a−/− neurons. Together with earlier analyses of Brn3 gene expression patterns in the retina and inner ear, these experiments suggest a deep functional similarity between primary somatosensory neurons, spiral and vestibular ganglion neurons, and retinal ganglion cells. This work also demonstrates the utility of sparse genetically-directed labeling for visualizing individual somatosensory afferent arbors and for defining cell-autonomous mutant phenotypes.
doi:10.1523/JNEUROSCI.4755-11.2012
PMCID: PMC3428801  PMID: 22262898
dorsal root ganglion; mechanoreceptor; skin; spinal cord; axonal arbor
10.  CLASS5 TRANSMEMBRANE SEMAPHORINS CONTROL SELECTIVE MAMMALIAN RETINAL LAMINATION AND FUNCTION 
Neuron  2011;71(3):460-473.
SUMMARY
In the vertebrate retina, neurites from distinct neuronal cell types are constrained within the plexiform layers, allowing for establishment of retinal lamination. However, the mechanisms by which retinal neurites are segregated within the inner or outer plexiform layers are not known. We find that the transmembrane semaphorins Sema5A and Sema5B constrain neurites from multiple retinal neuron subtypes within the inner plexiform layer (IPL). In Sema5A−/−; Sema5B−/− mice, retinal ganglion cells (RGCs), amacrine and bipolar cells exhibit severe defects leading to neurite mistargeting into the outer portions of the retina. These targeting abnormalities are more prominent in the outer (OFF) layers of the IPL and result in functional defects in select RGC response properties. Sema5A and Sema5B inhibit retinal neurite outgrowth through PlexinA1 and PlexinA3 receptors both in vitro and in vivo. These findings define a set of ligands and receptors required for the establishment of inner retinal lamination and function.
doi:10.1016/j.neuron.2011.06.009
PMCID: PMC3164552  PMID: 21835343
11.  An MRI-based Atlas and Database of the Developing Mouse Brain 
NeuroImage  2010;54(1):80-89.
The advent of mammalian gene engineering and genetically modified mouse models has led to renewed interest in developing resources for referencing and quantitative analysis of mouse brain anatomy. In this study, we used diffusion tensor imaging (DTI) for quantitative characterization of anatomical phenotypes in the developing mouse brain. As an anatomical reference for neuroscience research using mouse models, this paper presents DTI based atlases of ex vivo C57BL/6 mouse brains at several developmental stages. The atlas complements existing histology and MRI-based atlases by providing users access to three-dimensional, high-resolution images of the developing mouse brain, with distinct tissue contrasts and segmentations of major gray matter and white matter structures. The usefulness of the atlas and database was demonstrated by quantitative measurements of the development of major gray matter and white matter structures. Population average images of the mouse brain at several postnatal stages were created using large deformation diffeomorphic metric mapping and their anatomical variations were quantitatively characterized. The atlas and database enhance our ability to examine the neuroanatomy in normal or genetically engineered mouse strains and mouse models of neurological diseases.
doi:10.1016/j.neuroimage.2010.07.043
PMCID: PMC2962762  PMID: 20656042
12.  Preclinical assessment of CNS drug action using eye movements in mice 
The Journal of Clinical Investigation  2011;121(9):3528-3541.
The drug development process for CNS indications is hampered by a paucity of preclinical tests that accurately predict drug efficacy in humans. Here, we show that a wide variety of CNS-active drugs induce characteristic alterations in visual stimulus–induced and/or spontaneous eye movements in mice. Active compounds included sedatives and antipsychotic, antidepressant, and antiseizure drugs as well as drugs of abuse, such as cocaine, morphine, and phencyclidine. The use of quantitative eye-movement analysis was demonstrated by comparing it with the commonly used rotarod test of motor coordination and by using eye movements to monitor pharmacokinetics, blood-brain barrier penetration, drug-receptor interactions, heavy metal toxicity, pharmacologic treatment in a model of schizophrenia, and degenerative CNS disease. We conclude that eye-movement analysis could complement existing animal tests to improve preclinical drug development.
doi:10.1172/JCI45557
PMCID: PMC3163951  PMID: 21821912
13.  Morphologies of mouse retinal ganglion cells expressing transcription factors Brn3a, Brn3b, and Brn3c: analysis of wild type and mutant cells using genetically-directed sparse labeling 
Vision research  2010;51(2):269-279.
The mammalian retina contains more than 50 distinct neuronal types, which are broadly classified into several major classes: photoreceptor, bipolar, horizontal, amacrine, and ganglion cells. Although some of the developmental mechanisms involved in the differentiation of retinal ganglion cells (RGCs) are beginning to be understood, there is little information regarding the genetic and molecular determinants of the distinct morphologies of the 15 – 20 mammalian RGC cell types. Previous work has shown that the transcription factor Brn3b/Pou4f2 plays a major role in the development and survival of many RGCs. The roles of the closely related family members, Brn3a/Pou4f1 and Brn3c/Pou4f3 in RGC development are less clear. Using a genetically-directed method for sparse cell labeling and sparse conditional gene ablation in mice, we describe here the sets of RGC types in which each of the three Brn3/Pou4f transcription factors are expressed and the consequences of ablating these factors on the development of RGC morphologies.
doi:10.1016/j.visres.2010.08.039
PMCID: PMC3038626  PMID: 20826176
Mouse; Retina Development; Retinal Ganglion Cell; Brn3 Transcription Factors; Dendritic Arbor
14.  Expression of the Norrie disease gene (Ndp) in developing and adult mouse eye, ear, and brain 
Gene expression patterns : GEP  2010;11(1-2):151-155.
The Norrie disease gene (Ndp) codes for a secreted protein, Norrin, that activates canonical Wnt signaling by binding to its receptor, Frizzled-4. This signaling system is required for normal vascular development in the retina and for vascular survival in the cochlea. In mammals, the pattern of Ndp expression beyond the retina is poorly defined due to the low abundance of Norrin mRNA and protein. Here we characterize Ndp expression during mouse development by studying a knock-in mouse that carries the coding sequence of human placental alkaline phosphatase (AP) inserted at the Ndp locus (NdpAP). In the CNS, NdpAP expression is apparent by E10.5 and is dynamic and complex. The anatomically delimited regions of NdpAP expression observed prenatally in the CNS are replaced postnatally by widespread expression in astrocytes in the forebrain and midbrain, Bergman glia in the cerebellum, and Müller glia in the retina. In the developing and adult cochlea, NdpAP expression is closely associated with two densely vascularized regions, the stria vascularis and a capillary plexus between the organ of Corti and the spiral ganglion. These observations suggest the possibility that Norrin may have developmental and/or homeostatic functions beyond the retina and cochlea.
doi:10.1016/j.gep.2010.10.007
PMCID: PMC3061303  PMID: 21055480
Norrin; Norrie disease; Frizzled-4; mouse; brain development; vascular development
15.  The Norrin/Frizzled4 signaling pathway in retinal vascular development and disease 
Trends in molecular medicine  2010;16(9):417-425.
Disorders of retinal vascular growth and function are responsible for vision loss in a variety of diseases, including diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, and retinal artery or vein occlusion. Over the past decade, a new signaling pathway that controls retinal vascular development has emerged from the study of inherited disorders - in both humans and mice - that are characterized by retinal hypovascularization. This pathway utilizes a glial-derived extracellular ligand, Norrin, that acts on a transmembrane receptor, Frizzled4, a coreceptor, Lrp5, and an auxiliary membrane protein, Tspan12, on the surface of developing endothelial cells. The resulting signal controls a transcriptional program that regulates endothelial growth and maturation. It will be of great interest to determine whether modulating this pathway could represent a therapeutic approach to human retinal vascular disease.
doi:10.1016/j.molmed.2010.07.003
PMCID: PMC2963063  PMID: 20688566
16.  Structure-Function Analysis of the Bestrophin Family of Anion Channels* 
The Journal of biological chemistry  2003;278(42):41114-41125.
The bestrophins are a newly described family of anion channels unrelated in primary sequence to any previously characterized channel proteins. The human genome codes for four bestrophins, each of which confers a distinctive plasma membrane conductance on transfected 293 cells. Extracellular treatment with methanethiosulfonate ethyltrimethylammonium (MTSET) of a series of substitution mutants that eliminate one or more cysteines from human bestrophin1 demonstrates that cysteine 69 is the single endogenous cysteine responsible for MTSET inhibition of whole-cell current. Cysteines introduced between positions 78–99 and 223–226 are also accessible to external MTSET, with MTSET modification at positions 79, 80, 83, and 90 producing a 2–6-fold increase in whole-cell current. The latter set of four cysteine-substitution mutants define a region that appears to mediate allosteric control of channel activity. Mapping of transmembrane topography by insertion of N-linked glycosylation sites and tobacco etch virus protease cleavage sites provides evidence for cytosolic N and C termini and an unexpected transmembrane topography with at least three extracellular loops that include positions 60–63, 212–227, and 261–267. These experiments provide the first structural analysis of the bestrophin channel family.
doi:10.1074/jbc.M306150200
PMCID: PMC2885917  PMID: 12907679
17.  Norrin, Frizzled4, and Lrp5 signaling in endothelial cells controls a genetic program for retinal vascularization 
Cell  2009;139(2):285-298.
SUMMARY
Disorders of vascular structure and function play a central role in a wide variety of CNS diseases. Mutations in the Frizzled4 (Fz4) receptor, Lrp5 co-receptor, or Norrin ligand cause retinal hypovascularization, but the role of Norrin/Fz4/Lrp signaling in vascular development has not been defined. Using mouse genetic and cell culture models, we show that loss of Fz4 signaling in endothelial cells causes defective vascular growth, which leads to chronic but reversible silencing of retinal neurons. Loss of Fz4 in all endothelial cells disrupts the blood brain barrier in the cerebellum, while excessive Fz4 signaling disrupts embryonic angiogenesis. Sox17, a transcription factor that is up-regulated by Norrin/Fz4/Lrp signaling, plays a central role in inducing the angiogenic program controlled by Norrin/Fz4/Lrp. These experiments establish a cellular basis for retinal hypovascularization diseases due to insufficient Frizzled signaling, and they suggest a broader role for Frizzled signaling in vascular growth, remodeling, maintenance, and disease.
doi:10.1016/j.cell.2009.07.047
PMCID: PMC2779707  PMID: 19837032
18.  New Mouse Lines for the Analysis of Neuronal Morphology Using CreER(T)/loxP-Directed Sparse Labeling 
PLoS ONE  2009;4(11):e7859.
Background
Pharmacologic control of Cre-mediated recombination using tamoxifen-dependent activation of a Cre-estrogen receptor ligand binding domain fusion protein [CreER(T)] is widely used to modify and/or visualize cells in the mouse.
Methods and Findings
We describe here two new mouse lines, constructed by gene targeting to the Rosa26 locus to facilitate Cre-mediated cell modification. These lines should prove particularly useful in the context of sparse labeling experiments. The R26rtTACreER line provides ubiquitous expression of CreER under transcriptional control by the tetracycline reverse transactivator (rtTA); dual control by doxycycline and tamoxifen provides an extended dynamic range of Cre-mediated recombination activity. The R26IAP line provides high efficiency Cre-mediated activation of human placental alkaline phosphatase (hPLAP), complementing the widely used, but low efficiency, Z/AP line. By crossing with mouse lines that direct cell-type specific CreER expression, the R26IAP line has been used to produce atlases of labeled cholinergic and catecholaminergic neurons in the mouse brain. The R26IAP line has also been used to visualize the full morphologies of retinal dopaminergic amacrine cells, among the largest neurons in the mammalian retina.
Conclusions
The two new mouse lines described here expand the repertoire of genetically engineered mice available for controlled in vivo recombination and cell labeling using the Cre-lox system.
doi:10.1371/journal.pone.0007859
PMCID: PMC2775668  PMID: 19924248
19.  Distinct roles of transcription factors Brn3a and Brn3b in controlling the development, morphology, and function of retinal ganglion cells 
Neuron  2009;61(6):852-864.
SUMMARY
Transcriptional regulatory networks that control the morphologic and functional diversity of mammalian neurons are still largely undefined. Here we dissect the roles of the highly homologous POU-domain transcription factors Brn3a and Brn3b in retinal ganglion cell (RGC) development and function using conditional Brn3a and Brn3b alleles that permit the visualization of individual wild type or mutant cells. We show that Brn3a- and Brn3b-expressing RGCs exhibit overlapping but distinct dendritic stratifications and central projections. Deletion of Brn3a alters dendritic stratification and the ratio of monostratified: bistratified RGCs, with little or no change in central projections. In contrast, deletion of Brn3b leads to RGC transdifferentiation and loss, axon defects in the eye and brain, and defects in central projections that differentially compromise a variety of visually-driven behaviors. These findings reveal distinct roles for Brn3a and Brn3b in programming RGC diversity, and they illustrate the broad utility of germ-line methods for genetically manipulating and visualizing individual identified mammalian neurons.
doi:10.1016/j.neuron.2009.01.020
PMCID: PMC2679215  PMID: 19323995
20.  The genomic response of the retinal pigment epithelium to light damage and retinal detachment 
The retinal pigment epithelium (RPE) plays an essential role in maintaining the health of the retina. The RPE is also the site of pathologic processes in a wide variety of retinal disorders including monogenic retinal dystrophies, age-related macular degeneration, and retinal detachment. Despite intense interest in the RPE, little is known about its molecular response to ocular damage or disease. We have conducted a comprehensive analysis of changes in transcript abundance (the “genomic response”) in the murine RPE following light damage. Several dozen transcripts, many related to cell-cell signaling, show significant increases in abundance in response to bright light; transcripts encoding visual cycle proteins show a decrease in abundance. Similar changes are induced by retinal detachment. Environmental and genetic perturbations that modulate the RPE response to bright light suggest that this response is controlled by the retina. In contrast to the response to bright light, the RPE response to retinal detachment over-rides these modulatory affects.
doi:10.1523/JNEUROSCI.2401-08.2008
PMCID: PMC2570851  PMID: 18815272
retinal pigment epithelium; transcription; micro-array; mouse; ocular disease; light damage; retinal detachment
21.  Genetically-Directed, Cell Type-Specific Sparse Labeling for the Analysis of Neuronal Morphology 
PLoS ONE  2008;3(12):e4099.
Background
In mammals, genetically-directed cell labeling technologies have not yet been applied to the morphologic analysis of neurons with very large and complex arbors, an application that requires extremely sparse labeling and that is only rendered practical by limiting the labeled population to one or a few predetermined neuronal subtypes.
Methods and Findings
In the present study we have addressed this application by using CreER technology to non-invasively label very small numbers of neurons so that their morphologies can be fully visualized. Four lines of IRES-CreER knock-in mice were constructed to permit labeling selectively in cholinergic or catecholaminergic neurons [choline acetyltransferase (ChAT)-IRES-CreER or tyrosine hydroxylase (TH)-IRES-CreER], predominantly in projection neurons [neurofilament light chain (NFL)-IRES-CreER], or broadly in neurons and some glia [vesicle-associated membrane protein2 (VAMP2)-IRES-CreER]. When crossed to the Z/AP reporter and exposed to 4-hydroxytamoxifen in the early postnatal period, the number of neurons expressing the human placental alkaline phosphatase reporter can be reproducibly lowered to fewer than 50 per brain. Sparse Cre-mediated recombination in ChAT-IRES-CreER;Z/AP mice shows the full axonal and dendritic arbors of individual forebrain cholinergic neurons, the first time that the complete morphologies of these very large neurons have been revealed in any species.
Conclusions
Sparse genetically-directed, cell type-specific neuronal labeling with IRES-creER lines should prove useful for studying a wide variety of questions in neuronal development and disease.
doi:10.1371/journal.pone.0004099
PMCID: PMC2605552  PMID: 19116659
22.  The Optokinetic Reflex as a Tool for Quantitative Analyses of Nervous System Function in Mice: Application to Genetic and Drug-Induced Variation 
PLoS ONE  2008;3(4):e2055.
The optokinetic reflex (OKR), which serves to stabilize a moving image on the retina, is a behavioral response that has many favorable attributes as a test of CNS function. The OKR requires no training, assesses the function of diverse CNS circuits, can be induced repeatedly with minimal fatigue or adaptation, and produces an electronic record that is readily and objectively quantifiable.
We describe a new type of OKR test apparatus in which computer-controlled visual stimuli and streamlined data analysis facilitate a relatively high throughput behavioral assay. We used this apparatus, in conjunction with infrared imaging, to quantify basic OKR stimulus-response characteristics for C57BL/6J and 129/SvEv mouse strains and for genetically engineered lines lacking one or more photoreceptor systems or with an alteration in cone spectral sensitivity. A second generation (F2) cross shows that the characteristic difference in OKR frequency between C57BL/6J and 129/SvEv is inherited as a polygenic trait. Finally, we demonstrate the sensitivity and high temporal resolution of the OKR for quantitative analysis of CNS drug action.
These experiments show that the mouse OKR is well suited for neurologic testing in the context of drug discovery and large-scale phenotyping programs.
doi:10.1371/journal.pone.0002055
PMCID: PMC2323102  PMID: 18446207
23.  Ca2+-activated Cl− Current from Human Bestrophin-4 in Excised Membrane Patches 
The Journal of General Physiology  2006;127(6):749-754.
Bestrophins are a newly discovered family of Cl− channels, some members of which are activated by intracellular Ca2+. So far, all studies were carried out with whole-cell recordings from plasmid-transfected cultured cells, so it is unclear whether Ca2+ activates bestrophin through a metabolic mechanism or in a more direct way. We report here experiments that addressed this question with excised, inside-out membrane patches. We chose human bestrophin-4 (hBest4) for heterologous expression because it gave particularly large Cl− currents when expressed, thus allowing detection even in excised membrane patches. hBest4 gave a negligible Cl− current in a Ca2+-free solution on the cytoplasmic (bath) side, but produced a Cl− current that was activated by Ca2+ in a dose-dependent manner, with a K1/2 of 230 nM. Thus, Ca2+ appears to activate the bestrophin Cl− channel without going through a freely diffusible messenger or through protein phosphorylation. Because the activation and deactivation kinetics were very slow, however, we cannot exclude the involvement of a membrane-associated messenger.
doi:10.1085/jgp.200609527
PMCID: PMC2151534  PMID: 16702355
24.  Normal Light Response, Photoreceptor Integrity, and Rhodopsin Dephosphorylation in Mice Lacking Both Protein Phosphatases with EF Hands (PPEF-1 and PPEF-2) 
Molecular and Cellular Biology  2001;21(24):8605-8614.
Rhodopsin dephosphorylation in Drosophila is a calcium-dependent process that appears to be catalyzed by the protein product of the rdgC gene. Two vertebrate rdgC homologs, PPEF-1 and PPEF-2, have been identified. PPEF-1 transcripts are present at low levels in the retina, while PPEF-2 transcripts and PPEF-2 protein are abundant in photoreceptors. To determine if PPEF-2 alone or in combination with PPEF-1 plays a role in rhodopsin dephosphorylation and to determine if retinal degeneration accompanies mutation of PPEF-1 and/or PPEF-2, we have produced mice carrying targeted disruptions in the PPEF-1 and PPEF-2 genes. Loss of either or both PPEFs has little or no effect on rod function, as mice lacking both PPEF-1 and PPEF-2 show little or no changes in the electroretinogram and PPEF-2−/− mice show normal single-cell responses to light in suction pipette recordings. Light-dependent rhodopsin phosphorylation and dephosphorylation are also normal or nearly normal as determined by (i) immunostaining of PPEF-2−/− retinas with the phosphorhodopsin-specific antibody RT-97 and (ii) mass spectrometry of C-terminal rhodopsin peptides from mice lacking both PPEF-1 and PPEF-2. Finally, PPEF-2−/− retinas show normal histology at 1 year of age, and retinas from mice lacking both PPEF-1 and PPEF-2 show normal histology at 3 months of age, the latest time examined. These data indicate that, in contrast to loss of rdgC function in Drosophila, elimination of PPEF function does not cause retinal degeneration in vertebrates.
doi:10.1128/MCB.21.24.8605-8614.2001
PMCID: PMC100021  PMID: 11713293

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