Signaling Pathways in Neurovascular Development.

During development, the central nervous system (CNS) vasculature grows to precisely meet the metabolic demands of neurons and glia. In addition, the vast majority of the CNS vasculature acquires a unique set of molecular and cellular properties-collectively referred to as the blood-brain barrier-that minimize passive diffusion of molecules between the blood and the CNS parenchyma. Both of these processes are controlled by signals emanating from neurons and glia. In this review, we describe the nature and mechanisms-of-action of these signals, with an emphasis on vascular endothelial growth factor (VEGF) and beta-catenin (canonical Wnt) signaling, the two best-understood systems that regulate CNS vascular development. We highlight foundational discoveries, interactions between different signaling systems, the integration of genetic and cell biological studies, advances that are of clinical relevance, and questions for future research.

Norrin/Frizzled4 signaling in retinal vascular development and blood brain barrier plasticity.

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 intraretinal vascular architecture. In genetic mosaics, wild-type endothelial cells (ECs) instruct neighboring Fz4(-/-) ECs to produce an architecturally normal mosaic vasculature, a cell nonautonomous 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 and blood brain barrier function, indicating an ongoing requirement for Frizzled signaling in barrier maintenance and substantial plasticity in mature CNS vascular structure.

Normal and Sjogren's syndrome models of the murine lacrimal gland studied at single-cell resolution.

The lacrimal gland is of central interest in ophthalmology both as the source of the aqueous component of tear fluid and as the site of autoimmune pathology in the context of Sjogren's syndrome (SjS). To provide a foundational description of mouse lacrimal gland cell types and their patterns of gene expression, we have analyzed single-cell transcriptomes from wild-type (Balb/c) mice and from two genetically based SjS models, MRL/lpr and NOD (nonobese diabetic).H2b, and defined the localization of multiple cell-type-specific protein and mRNA markers. This analysis has uncovered a previously undescribed cell type, Car6+ cells, which are located at the junction of the acini and the connecting ducts. More than a dozen secreted polypeptides that are likely to be components of tear fluid are expressed by acinar cells and show pronounced sex differences in expression. Additional examples of gene expression heterogeneity within a single cell type were identified, including a gradient of Claudin4 along the length of the ductal system and cell-to-cell heterogeneity in transcription factor expression within acinar and myoepithelial cells. The patterns of expression of channels, transporters, and pumps in acinar, Car6+, and ductal cells make strong predictions regarding the mechanisms of water and electrolyte secretion. In MRL/lpr and NOD.H2b lacrimal glands, distinctive changes in parenchymal gene expression and in immune cell subsets reveal widespread interferon responses, a T cell-dominated infiltrate in the MRL/lpr model, and a mixed B cell and T cell infiltrate in the NOD.H2b model.

Utility of protein-protein binding surfaces composed of anti-parallel alpha-helices and beta-sheets selected by phage display.

Over the past 3 decades, a diverse collection of small protein domains have been used as scaffolds to generate general purpose protein-binding reagents using a variety of protein display and enrichment technologies. To expand the repertoire of scaffolds and protein surfaces that might serve this purpose, we have explored the utility of (i) a pair of anti-parallel alpha-helices in a small highly disulfide-bonded 4-helix bundle, the CC4 domain from reversion-inducing Cysteine-rich Protein with Kazal Motifs and (ii) a concave beta-sheet surface and two adjacent loops in the human FN3 domain, the scaffold for the widely used monobody platform. Using M13 phage display and next generation sequencing, we observe that, in both systems, libraries of ∼30 million variants contain binding proteins with affinities in the low µM range for baits corresponding to the extracellular domains of multiple mammalian proteins. CC4- and FN3-based binding proteins were fused to the N- and/or C-termini of Fc domains and used for immunostaining of transfected cells. Additionally, FN3-based binding proteins were inserted into VP1 of AAV to direct AAV infection to cells expressing a defined surface receptor. Finally, FN3-based binding proteins were inserted into the Pvc13 tail fiber protein of an extracellular contractile injection system particle to direct protein cargo delivery to cells expressing a defined surface receptor. These experiments support the utility of CC4 helices B and C and of FN3 beta-strands C, D, and F together with adjacent loops CD and FG as surfaces for engineering general purpose protein-binding reagents.

Hypoxia tolerance in the Norrin-deficient retina and the chronically hypoxic brain studied at single-cell resolution.

The mammalian CNS is capable of tolerating chronic hypoxia, but cell type-specific responses to this stress have not been systematically characterized. In the Norrin KO (NdpKO ) mouse, a model of familial exudative vitreoretinopathy (FEVR), developmental hypovascularization of the retina produces chronic hypoxia of inner nuclear-layer (INL) neurons and Muller glia. We used single-cell RNA sequencing, untargeted metabolomics, and metabolite labeling from 13C-glucose to compare WT and NdpKO retinas. In NdpKO retinas, we observe gene expression responses consistent with hypoxia in Muller glia and retinal neurons, and we find a metabolic shift that combines reduced flux through the TCA cycle with increased synthesis of serine, glycine, and glutathione. We also used single-cell RNA sequencing to compare the responses of individual cell types in NdpKO retinas with those in the hypoxic cerebral cortex of mice that were housed for 1 week in a reduced oxygen environment (7.5% oxygen). In the hypoxic cerebral cortex, glial transcriptome responses most closely resemble the response of Muller glia in the NdpKO retina. In both retina and brain, vascular endothelial cells activate a previously dormant tip cell gene expression program, which likely underlies the adaptive neoangiogenic response to chronic hypoxia. These analyses of retina and brain transcriptomes at single-cell resolution reveal both shared and cell type-specific changes in gene expression in response to chronic hypoxia, implying both shared and distinct cell type-specific physiologic responses.

Patterning of papillae on the mouse tongue: A system for the quantitative assessment of planar cell polarity signaling.

The dorsal surface of the mouse tongue is covered by ~7000 papillae, asymmetric epithelial protrusions that are precisely oriented to create a stereotyped macroscopic pattern. Within the context of this large-scale pattern, neighboring papillae exhibit a high degree of local order that minimizes the differences in their orientations. We show here that the orientations of lingual papillae are under the control of the core planar cell polarity (PCP) genes Vangl1, Vangl2, and Celsr1. Using K14-Cre and Nkx2.5-Cre to induce conditional knockout of Vangl1 and/or Vangl2 in the tongue epithelium, we observe more severe disruptions to local order among papillae with inactivation of larger numbers of Vangl genes, a greater role for Vangl2 than Vangl1, and a more severe phenotype with the Vangl2 Looptail (Lp) allele than the Vangl2 null allele, consistent with a dominant negative mode of action of the Vangl2Lp allele. Interestingly, Celsr1-/- tongues show disruption of both local and global order, with many papillae in the anterior tongue showing a reversed orientation. To quantify each of these phenotypes, we have developed and applied three procedures for sampling the orientations of papillae and assessing the degree of order on different spatial scales. The experiments reported here establish the dorsal surface of the mouse tongue as a favorable system for studying PCP control of epithelial patterning.

Endothelin-2 signaling in the neural retina promotes the endothelial tip cell state and inhibits angiogenesis.

Endothelin signaling is required for neural crest migration and homeostatic regulation of blood pressure. Here, we report that constitutive overexpression of Endothelin-2 (Edn2) in the mouse retina perturbs vascular development by inhibiting endothelial cell migration across the retinal surface and subsequent endothelial cell invasion into the retina. Developing endothelial cells exist in one of two states: tip cells at the growing front and stalk cells in the vascular plexus behind the front. This division of endothelial cell states is one of the central organizing principles of angiogenesis. In the developing retina, Edn2 overexpression leads to overproduction of endothelial tip cells by both morphologic and molecular criteria. Spatially localized overexpression of Edn2 produces a correspondingly localized endothelial response. Edn2 overexpression in the early embryo inhibits vascular development at midgestation, but Edn2 overexpression in developing skin and brain has no discernible effect on vascular structure. Inhibition of retinal angiogenesis by Edn2 requires expression of Endothelin receptor A but not Endothelin receptor B in the neural retina. Taken together, these observations imply that the neural retina responds to Edn2 by synthesizing one or more factors that promote the endothelial tip cell state and inhibit angiogenesis. The response to Edn2 is sufficiently potent that it overrides the activities of other homeostatic regulators of angiogenesis, such as Vegf.

Injury-independent induction of reactive gliosis in retina by loss of function of the LIM homeodomain transcription factor Lhx2.

Müller glia are the primary glial subtype in the retina and perform a wide range of physiological tasks in support of retinal function, but little is known about the transcriptional network that maintains these cells in their differentiated state. We report that selective deletion of the LIM homeodomain transcription factor Lhx2 from mature Müller glia leads to the induction of reactive retinal gliosis in the absence of injury. Furthermore, Lhx2 expression is also down-regulated in Prph2(Rd2/Rd2) animals immediately before the onset of reactive gliosis. Analysis of conditional Lhx2 knockouts showed that gliosis was hypertrophic but not proliferative. Aging of experimental animals demonstrated that constitutive reactive gliosis induced by deletion of Lhx2 reduced rates of ongoing apoptosis and compromised both rod and cone photoreceptor function. Additionally, these animals showed a dramatically reduced ability to induce expression of secreted neuroprotective factors and displayed enhanced rates of apoptosis in light-damage assays. We provide in vivo evidence that Lhx2 actively maintains mature Müller glia in a nonreactive state, with loss of function initiating a specific program of nonproliferative hypertrophic gliosis.

Genetic mosaic analysis reveals a major role for frizzled 4 and frizzled 8 in controlling ureteric growth in the developing kidney.

The developing mammalian kidney is an attractive system in which to study the control of organ growth. Targeted mutations in the Wnt receptors frizzled (Fz) 4 and Fz8 lead to reduced ureteric bud growth and a reduction in kidney size, a phenotype previously reported for loss of Wnt11. In cell culture, Fz4 and Fz8 can mediate noncanonical signaling stimulated by Wnt11, but only Fz4 mediates Wnt11-stimulated canonical signaling. In genetically mosaic mouse ureteric buds, competition between phenotypically mutant Fz4(-/-) or Fz4(-/-);Fz8(-/-) cells and adjacent phenotypically wild-type Fz4(+/-) or Fz4(+/-);Fz8(-/-) cells results in under-representation of the mutant cells to an extent far greater than would be predicted from the size reduction of homogeneously mutant kidneys. This discrepancy presumably reflects the compensatory action of a network of growth regulatory systems that minimize developmental perturbations. The present work represents the first description of a kidney phenotype referable to one or more Wnt receptors and demonstrates a general strategy for revealing the contribution of an individual growth regulatory pathway when it is part of a larger homeostatic network.

Bacterial meningitis in the early postnatal mouse studied at single-cell resolution.

Bacterial meningitis is a major cause of morbidity and mortality, especially among infants and the elderly. Here, we study mice to assess the response of each of the major meningeal cell types to early postnatal E. coli infection using single nucleus RNA sequencing (snRNAseq), immunostaining, and genetic and pharamacologic perturbations of immune cells and immune signaling. Flatmounts of the dissected leptomeninges and dura were used to facilitiate high-quality confocal imaging and quantification of cell abundances and morphologies. Upon infection, the major meningeal cell types - including endothelial cells (ECs), macrophages, and fibroblasts - exhibit distinctive changes in their transcriptomes. Additionally, ECs in the leptomeninges redistribute CLDN5 and PECAM1, and leptomeningeal capillaries exhibit foci with reduced blood-brain barrier integrity. The vascular response to infection appears to be largely driven by TLR4 signaling, as determined by the nearly identical responses induced by infection and LPS administration and by the blunted response to infection in Tlr4-/- mice. Interestingly, knocking out Ccr2, encoding a major chemoattractant for monocytes, or acute depletion of leptomeningeal macrophages, following intracebroventricular injection of liposomal clodronate, had little or no effect on the response of leptomeningeal ECs to E. coli infection. Taken together, these data imply that EC responses to infection are largely driven by the intrinsic EC response to LPS.

A transcriptome atlas of the mouse iris at single-cell resolution defines cell types and the genomic response to pupil dilation.

The iris controls the level of retinal illumination by controlling pupil diameter. It is a site of diverse ophthalmologic diseases and it is a potential source of cells for ocular auto-transplantation. The present study provides foundational data on the mouse iris based on single nucleus RNA sequencing. More specifically, this work has (1) defined all of the major cell types in the mouse iris and ciliary body, (2) led to the discovery of two types of iris stromal cells and two types of iris sphincter cells, (3) revealed the differences in cell type-specific transcriptomes in the resting vs. dilated states, and (4) identified and validated antibody and in situ hybridization probes that can be used to visualize the major iris cell types. By immunostaining for specific iris cell types, we have observed and quantified distortions in nuclear morphology associated with iris dilation and clarified the neural crest contribution to the iris by showing that Wnt1-Cre-expressing progenitors contribute to nearly all iris cell types, whereas Sox10-Cre-expressing progenitors contribute only to stromal cells. This work should be useful as a point of reference for investigations of iris development, disease, and pharmacology, for the isolation and propagation of defined iris cell types, and for iris cell engineering and transplantation.

Developmental, cellular, and behavioral phenotypes in a mouse model of congenital hypoplasia of the dentate gyrus.

In the hippocampus, a widely accepted model posits that the dentate gyrus improves learning and memory by enhancing discrimination between inputs. To test this model, we studied conditional knockout mice in which the vast majority of dentate granule cells (DGCs) fail to develop - including nearly all DGCs in the dorsal hippocampus - secondary to eliminating Wntless (Wls) in a subset of cortical progenitors with Gfap-Cre. Other cells in the Wlsfl/-;Gfap-Cre hippocampus were minimally affected, as determined by single nucleus RNA sequencing. CA3 pyramidal cells, the targets of DGC-derived mossy fibers, exhibited normal morphologies with a small reduction in the numbers of synaptic spines. Wlsfl/-;Gfap-Cre mice have a modest performance decrement in several complex spatial tasks, including active place avoidance. They were also modestly impaired in one simpler spatial task, finding a visible platform in the Morris water maze. These experiments support a role for DGCs in enhancing spatial learning and memory.

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 after 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 overrides these modulatory affects.

Roles of HIFs and VEGF in angiogenesis in the retina and brain.

Vascular development in the mammalian retina is a paradigm for CNS vascular development in general, and its study is revealing fundamental mechanisms that explain the efficacy of antiangiogenic therapies in retinal vascular disease. During development of the mammalian retina, hypoxic astrocytes are hypothesized to secrete VEGF, which attracts growing endothelial cells as they migrate radially from the optic disc. However, published tests of this model using astrocyte-specific deletion of Vegf in the developing mouse retina appear to contradict this theory. Here, we report that selectively eliminating Vegf in neonatal retinal astrocytes with a Gfap-Cre line that recombines with approximately 100% efficiency had no effect on proliferation or radial migration of astrocytes, but completely blocked radial migration of endothelial cells, strongly supporting the hypoxic astrocyte model. Using additional Cre driver lines, we found evidence for essential and partially redundant actions of retina-derived (paracrine) and astrocyte-derived (autocrine) VEGF in controlling astrocyte proliferation and migration. We also extended previous studies by showing that HIF-1α in retinal neurons and HIF-2α in Müller glia play distinct roles in retinal vascular development and disease, adding to a growing body of data that point to the specialization of these 2 hypoxia-sensing transcription factors.

Beta-catenin signaling regulates barrier-specific gene expression in circumventricular organ and ocular vasculatures.

The brain, spinal cord, and retina are supplied by capillaries that do not permit free diffusion of molecules between serum and parenchyma, a property that defines the blood-brain and blood-retina barriers. Exceptions to this pattern are found in circumventricular organs (CVOs), small midline brain structures that are supplied by high permeability capillaries. In the eye and brain, high permeability capillaries are also present in the choriocapillaris, which supplies the retinal pigment epithelium and photoreceptors, and the ciliary body and choroid plexus, the sources of aqueous humor and cerebrospinal fluid, respectively. We show here that (1) endothelial cells in these high permeability vascular systems have very low beta-catenin signaling compared to barrier-competent endothelial cells, and (2) elevating beta-catenin signaling leads to a partial conversion of permeable endothelial cells to a barrier-type state. In one CVO, the area postrema, high permeability is maintained, in part, by local production of Wnt inhibitory factor-1.

Affinity capture of polyribosomes followed by RNAseq (ACAPseq), a discovery platform for protein-protein interactions.

Defining protein-protein interactions (PPIs) is central to the biological sciences. Here, we present a novel platform - Affinity Capture of Polyribosomes followed by RNA sequencing (ACAPseq) - for identifying PPIs. ACAPseq harnesses the power of massively parallel RNA sequencing (RNAseq) to quantify the enrichment of polyribosomes based on the affinity of their associated nascent polypeptides for an immobilized protein 'bait'. This method was developed and tested using neonatal mouse brain polyribosomes and a variety of extracellular domains as baits. Of 92 baits tested, 25 identified one or more binding partners that appear to be biologically relevant; additional candidate partners remain to be validated. ACAPseq can detect binding to targets that are present at less than 1 part in 100,000 in the starting polyribosome preparation. One of the observed PPIs was analyzed in detail, revealing the mode of homophilic binding for Protocadherin-9 (PCDH9), a non-clustered Protocadherin family member.

Transcriptional and epigenomic landscapes of CNS and non-CNS vascular endothelial cells.

Vascular endothelial cell (EC) function depends on appropriate organ-specific molecular and cellular specializations. To explore genomic mechanisms that control this specialization, we have analyzed and compared the transcriptome, accessible chromatin, and DNA methylome landscapes from mouse brain, liver, lung, and kidney ECs. Analysis of transcription factor (TF) gene expression and TF motifs at candidate cis-regulatory elements reveals both shared and organ-specific EC regulatory networks. In the embryo, only those ECs that are adjacent to or within the central nervous system (CNS) exhibit canonical Wnt signaling, which correlates precisely with blood-brain barrier (BBB) differentiation and Zic3 expression. In the early postnatal brain, single-cell RNA-seq of purified ECs reveals (1) close relationships between veins and mitotic cells and between arteries and tip cells, (2) a division of capillary ECs into vein-like and artery-like classes, and (3) new endothelial subtype markers, including new validated tip cell markers.

The genomic response to retinal disease and injury: evidence for endothelin signaling from photoreceptors to glia.

Regardless of proximal cause, photoreceptor injury or disease almost invariably leads to the activation of Muller cells, the principal glial cells in the retina. This observation implies the existence of signaling systems that inform Muller cells of the health status of photoreceptors. It further suggests that diverse types of photoreceptor damage elicit a limited range of biochemical responses. Using the mouse retina, we show by microarray, RNA blot, and in situ hybridization that the genomic responses to both light damage and inherited photoreceptor degeneration involve a relatively small number of genes and that the genes activated by these two insults overlap substantially with one another and with the genes activated by retinal detachment. Among the induced transcripts, those coding for endothelin2 (Edn2) are unusual in that they are localized to photoreceptors and are also highly induced in all of the tested models of photoreceptor disease or injury. Acute light damage also leads to a >10-fold increase in endothelin receptor B (Ednrb) in Muller cells 24 h after injury. These observations suggest that photoreceptor-derived EDN2 functions as a general stress signal, that EDN2 signals to Muller cells by binding to EDNRB, and that Muller cells can increase their sensitivity to EDN2 as part of the injury response.

The rod photoreceptor-specific nuclear receptor Nr2e3 represses transcription of multiple cone-specific genes.

This study addresses one genetic regulatory mechanism that establishes the distinct identities of rod and cone photoreceptors. Previous work has shown that mutations in either humans or mice in the gene coding for photoreceptor-specific nuclear receptor Nr2e3 cause a progressive retinal degeneration characterized by increased numbers of short-wave cones. In the present work, we have examined the cellular and developmental pattern of Nr2e3 protein localization in mammals and fish, identified an optimal Nr2e3 DNA-binding site using cycles of binding to recombinant Nr2e3, characterized the transcriptional activity of wild type and one of the disease-associated point mutations in Nr2e3 in transfected cells, and characterized the transcriptional defects in the naturally occurring Nr2e3 mutant (rd7) mouse. These experiments indicate that in the mature vertebrate retina Nr2e3 is expressed exclusively in rods, that expression of Nr2e3 is one of the earliest events in the pathway of rod-specific photoreceptor development, and that Nr2e3 functions, either directly or indirectly, as a repressor of cone-specific genes in rod photoreceptor cells.

An evolutionary perspective on the photoreceptor damage response.

PURPOSE: To review recent advances related to the response to photoreceptor damage and to place this knowledge in an evolutionary context. DESIGN: Synthesis of published laboratory, clinical, and epidemiologic data. METHODS: The authors have synthesized the principal published findings related to the mechanism and function of the photoreceptor damage response with the goal of trying to understand the selective pressures that shaped its evolution. RESULTS: The past several years have seen considerable advances in understanding the molecular and cellular basis of the retina's response to photoreceptor damage. From their analysis of laboratory, clinical, and epidemiologic data, the authors suggest that the photoreceptor damage response may have evolved to counter the effects of retinal tears and detachment, infectious retinitis, and/or light damage. CONCLUSIONS: If the natural response to photoreceptor damage can be fully defined, it may be possible to augment desirable aspects of the damage response and/or suppress undesirable ones in the context of a wide variety of photoreceptor diseases.