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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.
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.
PMCID: PMC3950970  PMID: 24411735
3.  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.
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.
PMCID: PMC3865743  PMID: 24347548
planar cell polarity (PCP); axon growth; limb innervation; neural crest; cell death; muscle atrophy; Mouse
4.  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.
PMCID: PMC3535266  PMID: 23217714
5.  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.
PMCID: PMC3428801  PMID: 22262898
dorsal root ganglion; mechanoreceptor; skin; spinal cord; axonal arbor
6.  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.
PMCID: PMC3061303  PMID: 21055480
Norrin; Norrie disease; Frizzled-4; mouse; brain development; vascular development
7.  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.
PMCID: PMC2885917  PMID: 12907679
8.  Norrin, Frizzled4, and Lrp5 signaling in endothelial cells controls a genetic program for retinal vascularization 
Cell  2009;139(2):285-298.
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.
PMCID: PMC2779707  PMID: 19837032
9.  New Mouse Lines for the Analysis of Neuronal Morphology Using CreER(T)/loxP-Directed Sparse Labeling 
PLoS ONE  2009;4(11):e7859.
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.
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.
PMCID: PMC2775668  PMID: 19924248
10.  Genetically-Directed, Cell Type-Specific Sparse Labeling for the Analysis of Neuronal Morphology 
PLoS ONE  2008;3(12):e4099.
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.
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.
PMCID: PMC2605552  PMID: 19116659

Results 1-10 (10)