The expression and function of netrin-4 in murine ocular tissues.

Netrin-4, a member of the netrin family, is a potent regulator of embryonic development. It promotes neurite extension and regulates pulmonary airway branching, vasculogenesis patterning, and endothelial proliferation in pathological angiogenesis. The initial characterization of netrin-4 expression was focused on epithelial-derived organs (kidney, lung and salivary gland) and the central nervous system. Ocular development is an ideal system to study netrin-4 expression and function, as it involves both ectodermal (cornea, lens and retina) and mesodermal (sclera and choroid) derivatives and has an extensive and well-characterized angiogenic process. Netrin-4 is expressed in all ocular tissues. It is a prominent component of the basement membranes of the lens and cornea, as well as all three basement membranes of the retina: the inner limiting membrane, vascular basement membranes, and Bruch's membrane. Netrin-4 is differentially deposited in vascular basement membranes, with more intense anti-netrin-4 reactivity on the arterial side. The retinal microcirculation also expresses netrin-4. In order to test the function of netrin-4 in vivo, we generated a conventional mouse lacking Ntn4 expression. Basement membrane formation in the cornea, lens and retina is undisrupted by netrin-4 deletion, demonstrating that netrin-4 is not a major structural component of these basement membranes. In the Ntn4 homozygous null (Ntn4-/-) cornea, the overall morphology of the cornea, as well as the epithelial, stromal and endothelial stratification are normal; however, epithelial cell proliferation is increased. In the Ntn4-/- retina, neurogenesis appears to proceed normally, as does retinal lamination. In the Ntn4-/- retina, retinal ganglion cell targeting is intact, although there are minor defects in axon fasciculation. In the retinal vasculature of the Ntn4-/- retina, the distribution patterns of astrocytes and the vasculature are largely normal, with the possible exception of increased branching in the deep capillary plexus, suggesting that netrin-4 may act as a negative regulator of angiogenesis. These data, taken together, suggest that netrin-4 is a negative regulator of corneal epithelial cell proliferation and retinal vascular branching in vivo, whereas netrin-4 may be redundant with other members of the netrin family in other ocular tissue development. Ntn4-/- mice may serve as a good model in which to study the role of netrins in vivo of the pathobiologic vascular remodeling in the retina and cornea.

ß2 and γ3 laminins are critical cortical basement membrane components: ablation of Lamb2 and Lamc3 genes disrupts cortical lamination and produces dysplasia.

Cortical development is dependent on the timely production and migration of neurons from neurogenic sites to their mature positions. Mutations in several receptors for extracellular matrix (ECM) molecules and their downstream signaling cascades produce dysplasia in brain. Although mutation of a critical binding site in the gene that encodes the ECM molecule laminin γ1 (Lamc1) disrupts cortical lamination, the ECM ligand(s) for many ECM receptors have not been demonstrated directly in the cortex. Several isoforms of the heterotrimeric laminins, all containing the ß2 and γ3 chain, have been isolated from the brain, suggesting they are important for CNS function. Here, we report that mice homozygous null for the laminin ß2 and γ3 chains exhibit cortical laminar disorganization. Mice lacking both of these laminin chains exhibit hallmarks of human cobblestone lissencephaly (type II, nonclassical): they demonstrate severe laminar disruption; midline fusion; perturbation of Cajal-Retzius cell distribution; altered radial glial cell morphology; and ectopic germinal zones. Surprisingly, heterozygous mice also exhibit laminar disruption of cortical neurons, albeit with lesser severity. In compound null mice, the pial basement membrane is fractured, and the distribution of a key laminin receptor, dystroglycan, is altered. These data suggest that ß2 and γ3-containing laminins play an important dose-dependent role in development of the cortical pial basement membrane, which serves as an attachment site for Cajal-Retzius and radial glial cells, thereby guiding neural development.

Bipolar cell-photoreceptor connectivity in the zebrafish (Danio rerio) retina.

Bipolar cells convey luminance, spatial, and color information from photoreceptors to amacrine and ganglion cells. We studied the photoreceptor connectivity of 321 bipolar cells in the adult zebrafish retina. 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was inserted into whole-mounted transgenic zebrafish retinas to label bipolar cells. The photoreceptors that connect to these DiI-labeled cells were identified by transgenic fluorescence or their positions relative to the fluorescent cones, as cones are arranged in a highly ordered mosaic: rows of alternating blue- (B) and ultraviolet-sensitive (UV) single cones alternate with rows of red-(R) and green-sensitive (G) double cones. Rod terminals intersperse among cone terminals. As many as 18 connectivity subtypes were observed, 9 of which-G, GBUV, RG, RGB, RGBUV, RGRod, RGBRod, RGBUVRod, and RRod bipolar cells-accounted for 96% of the population. Based on their axon terminal stratification, these bipolar cells could be further subdivided into ON, OFF, and ON-OFF cells. The dendritic spread size, soma depth and size, and photoreceptor connections of the 308 bipolar cells within the nine common connectivity subtypes were determined, and their dendritic tree morphologies and axonal stratification patterns compared. We found that bipolar cells with the same axonal stratification patterns could have heterogeneous photoreceptor connectivity whereas bipolar cells with the same dendritic tree morphology usually had the same photoreceptor connectivity, although their axons might stratify on different levels.

The γ3 chain of laminin is widely but differentially expressed in murine basement membranes: expression and functional studies.

Laminins are heterotrimeric extracellular glycoproteins found in, but not confined to, basement membranes (BMs). They are important components in formation of the molecular networks of BMs as well as in cell polarity, cell differentiation and tissue morphogenesis. Each laminin is composed by an α, a ß and a γ chain. Previous studies have shown that the γ3 chain is partnered with either the ß1 chain (in placenta) or ß2 chain (in the CNS) (Libby et al., 2000). Several studies, including our own, suggested that the γ3 chain is expressed in both apical and basal compartments (Koch et al., 1999; Gersdorff et al., 2005; Yan and Cheng, 2006). This study investigates the expression pattern of the γ3 chain in mouse. We developed three new γ3-reactive antibodies, and we show that the γ3 chain is present in BMs. The distribution pattern is considerably more restricted than that of the γ1 chain and within any tissue there is differential deposition into BM compartments. This is particularly true in the retina and brain, where γ3 is uniquely expressed in a subset of the vascular basement membranes and the pial surface. We used conventional genetic ablation techniques to remove the γ3 chain in mice; unlike other laminin null mice (α5, ß2, γ1 nulls), these mice live a normal lifespan and have only minor abnormalities, the most striking of which are ectopic granule cells in the cerebellum and an apparent increase in capillary branching in the outer retina. These data support the suggestion that the γ3 chain is deposited in BMs and contributes some unique properties to their function, particularly in the nervous system.

Defective formation of the inner limiting membrane in laminin beta2- and gamma3-null mice produces retinal dysplasia.

PURPOSE: Retinal basement membranes (BMs) serve as attachment sites for retinal pigment epithelial cells on Bruch's membrane and Müller cells (MCs) on the inner limiting membrane (ILM), providing polarity cues to adherent cells. The beta2 and gamma3 chains of laminin are key components of retinal BMs throughout development, suggesting that they play key roles in retinal histogenesis. This study was conducted to analyze how the absence of both beta2- and gamma3-containing laminins affects retinal development. Methods. The function of the beta2- and gamma3-containing laminins was tested by producing a compound deletion of both the beta2 and the gamma3 laminin genes in the mouse and assaying the effect on postnatal retinal development by using anatomic and electrophysiological techniques. Results. Despite the widespread expression of beta2 and gamma3 laminin chains in wild-type (WT) retinal BMs, the development of only one, the ILM, was disrupted. The postnatal consequence of the ILM disruption was an alteration of MC attachment and a resultant disruption in MC apical-basal polarity, which culminated in retinal dysplasia. Of importance, although their density was altered, retinal cell fates were unaffected. The laminin mutants have a markedly decreased visual function, resulting in part from photoreceptor dysgenesis. Conclusions. These data suggest that beta2 and gamma3 laminin isoforms are critical for the formation and stability of the ILM. These data also suggest that attachment of the MC to the ILM provides important polarity cues to the MC and for postnatal retinal histogenesis.

Specificity of the horizontal cell-photoreceptor connections in the zebrafish (Danio rerio) retina.

Horizontal cells (HCs) are involved in establishing the center-surround receptive field organization of photoreceptor and bipolar cells. In many species, HCs respond differentially to colors and may play a role in color vision. An earlier study from our laboratory suggested that four types of HCs exist in the zebrafish retina: three cone HCs (H1, H2 and H3) and one rod HC. In this study, we describe their photoreceptor connections. Cones are arranged in a mosaic in which rows of alternating blue (B)- and ultraviolet (UV)-sensitive single cones alternate with rows of red (R)- and green (G)-sensitive double cones; the G cones are adjacent to UV cones and B cones adjacent to R cones. Two small-field (H1 and H2) and two large-field (H3 and rod HC) cells were observed. The cone HC dendritic terminals connected to cones with single boutons, doublets, or rosettes, whereas the rod HCs connected to rods with single boutons. The single boutons/doublets/rosettes of cone HCs were arranged in double rows separated by single rows for H1 cells, in pairs and singles for H2 cells, and in a rectilinear pattern for H3 cells. These connectivity patterns suggest that H1 cells contact R, G, and B cones, H2 cells G, B, and UV cones, and H3 cells B and UV cones. These predictions were confirmed by applying the DiI method to SWS1-GFP retinas whose UV cones express green fluorescent protein. Each rod HC was adjacent to the soma or axon of a DiI-labeled cone HC and connected to 50-200 rods.