Defects in the outer limiting membrane are associated with rosette development in the Nrl-/- retina

The neural retinal leucine zipper (Nrl) knockout mouse is a widely used model to study cone photoreceptor development, physiology, and molecular biology in the absence of rods. In the Nrl(-/-) retina, rods are converted into functional cone-like cells. The Nrl(-/-) retina is characterized by large undulations of the outer nuclear layer (ONL) commonly known as rosettes. Here we explore the mechanism of rosette development in the Nrl(-/-) retina. We report that rosettes first appear at postnatal day (P)8, and that the structure of nascent rosettes is morphologically distinct from what is seen in the adult retina. The lumen of these nascent rosettes contains a population of aberrant cells protruding into the subretinal space that induce infolding of the ONL. Morphologically adult rosettes do not contain any cell bodies and are first detected at P15. The cells found in nascent rosettes are photoreceptors in origin but lack inner and outer segments. We show that the adherens junctions between photoreceptors and Müller glia which comprise the retinal outer limiting membrane (OLM) are not uniformly formed in the Nrl(-/-) retina and thus allow protrusion of a population of developing photoreceptors into the subretinal space where their maturation becomes delayed. These data suggest that the rosettes of the Nrl(-/-) retina arise due to defects in the OLM and delayed maturation of a subset of photoreceptors, and that rods may play an important role in the proper formation of the OLM.     

Rod phosphodiesterase-6 (PDE6) catalytic subunits restore cone function in a mouse model lacking cone PDE6 catalytic subunit

Rod and cone photoreceptor neurons utilize discrete PDE6 enzymes that are crucial for phototransduction. Rod PDE6 is composed of heterodimeric catalytic subunits (αβ), while the catalytic core of cone PDE6 (α') is a homodimer. It is not known if variations between PDE6 subunits preclude rod PDE6 catalytic subunits from coupling to the cone phototransduction pathway. To study this issue, we generated a cone-dominated mouse model lacking cone PDE6 (Nrl(-/-) cpfl1). In this animal model, using several independent experimental approaches, we demonstrated the expression of rod PDE6 (αβ) and the absence of cone PDE6 (α') catalytic subunits. The rod PDE6 enzyme expressed in cone cells is active and contributes to the hydrolysis of cGMP in response to light. In addition, rod PDE6 expressed in cone cells couples to the light signaling pathway to produce S-cone responses. However, S-cone responses and light-dependent cGMP hydrolysis were eliminated when the β-subunit of rod PDE6 was removed (Nrl(-/-) cpfl1 rd). We conclude that either rod or cone PDE6 can effectively couple to the cone phototransduction pathway to mediate visual signaling. Interestingly, we also found that functional PDE6 is required for trafficking of M-opsin to cone outer segments.     

Nrl‐Cre transgenic mouse mediates loxP recombination in developing rod photoreceptors

The developing mouse retina is a tractable model for studying neurogenesis and differentiation. Although transgenic Cre mouse lines exist to mediate conditional genetic manipulations in developing mouse retinas, none of them act specifically in early developing rods. For conditional genetic manipulations of developing retinas, a Nrl‐Cre mouse line in which the Nrl promoter drives expression of Cre in rod precursors was created. The results showed that Nrl‐Cre expression was specific to the retina where it drives rod‐specific recombination with a temporal pattern similar to endogenous Nrl expression during retinal development. This Nrl‐Cre transgene does not negatively impact retinal structure and function. Taken together, the data suggested that the Nrl‐Cre mouse line was a valuable tool to drive Cre‐mediated recombination specifically in developing rods. genesis 54:129–135, 2016. © 2016 Wiley Periodicals, Inc.     

Increased cone sensitivity to ABCA4 deficiency provides insight into macular vision loss in Stargardt's dystrophy

Autosomal recessive Stargardt macular dystrophy is caused by mutations in the photoreceptor disc rim protein ABCA4/ABCR. Key clinical features of Stargardt disease include relatively mild rod defects such as delayed dark adaptation, coupled with severe cone defects reflected in macular atrophy and central vision loss. In spite of this clinical divergence, there has been no biochemical study of the effects of ABCA4 deficiency on cones vs. rods. Here we utilize the cone-dominant Abca4(-/-)/Nrl(-/-) double knockout mouse to study this issue. We show that as early as post-natal day (P) 30, Abca4(-/-)/Nrl(-/-) retinas have significantly fewer rosettes than Abca4(+/+)/Nrl(-/-) retinas, a phenotype often associated with accelerated degeneration. Abca4-deficient mice in both the wild-type and cone-dominant background accumulate more of the toxic bisretinoid A2E than their ABCA4-competent counterparts, but Abca4(-/-)/Nrl(-/-) eyes generate significantly more A2E per mole of 11-cis-retinal (11-cisRAL) than Abca4(-/-) eyes. At P120, Abca4(-/-)/Nrl(-/-) produced 340 ± 121 pmoles A2E/nmol 11-cisRAL while Abca4(-/-) produced 50.4 ± 8.05 pmoles A2E/nmol 11-cisRAL. Nevertheless, the retinal pigment epithelium (RPE) of Abca4(-/-)/Nrl(-/-) eyes exhibits fewer lipofuscin granules than the RPE of Abca4(-/-) eyes; at P120: Abca4(-/-)/Nrl(-/-) exhibit 0.045 ± 0.013 lipofuscingranules/μm2 of RPE vs. Abca4(-/-) 0.17 ± 0.030 lipofuscingranules/μm2 of RPE. These data indicate that ABCA4-deficient cones simultaneously generate more A2E than rods and are less able to effectively clear it, and suggest that primary cone toxicity may contribute to Stargardt's-associated macular vision loss in addition to cone death secondary to RPE atrophy.     

Photoreceptor topography in the duplex retina of the paddlefish (Polyodon spathula)

Retinal whole-mount preparations from the eyes of the North American paddlefish, Polyodon spathula, were examined with a combination of bright field and differential interference contrast microscopy. The entire retina was mapped and population counts of rod and cone photoreceptors were made at regular intervals throughout the retina. The retina is dominated by rods, but a significant percentage (ca. 38%) of the photoreceptors are cones. Mean cone packing density for the entire retina is 6,402+/-1,216 cones/mm2. There is a small (16%) but statistically significant difference between cone packing density in the dorsal retina (6,674+/-1,168 cones/mm2) and the ventral retina (5,745+/-1,076 cones/mm2). There is no region of unusually high cone concentration that might be construed as a fovea or a visual streak. Mean rod packing density for the entire retina is 10,271+/-1,205 rods/mm2. Except in the far periphery, where rods are less numerous, the density of rods is fairly uniform throughout the retina. The data are discussed with regard to paddlefish habitat and behavior.     

Inactivation of the dual Bmp/Wnt inhibitor Sostdc1 enhances pancreatic islet function

Current endeavors in the type 2 diabetes (T2D) field include gaining a better understanding of extracellular signaling pathways that regulate pancreatic islet function. Recent data suggest that both Bmp and Wnt pathways are operative in pancreatic islets and play a positive role in insulin secretion and glucose homeostasis. Our laboratory found the dual Bmp and Wnt antagonist Sostdc1 to be upregulated in a mouse model of islet dysmorphogenesis and nonimmune-mediated lean diabetes. Because Bmp signaling has been proposed to enhance β-cell function, we evaluated the role of Sostdc1 in adult islet function using animals in which Sostdc1 was globally deleted. While Sostdc1-null animals exhibited no pancreas development phenotype, a subset of mutants exhibited enhanced insulin secretion and improved glucose homeostasis compared with control animals after 12-wk exposure to high-fat diet. Loss of Sostdc1 in the setting of metabolic stress results in altered expression of Bmp-responsive genes in islets but did not affect expression of Wnt target genes, suggesting that Sostdc1 primarily regulates the Bmp pathway in the murine pancreas. Furthermore, our data indicate that removal of Sostdc1 enhances the downregulation of the closely related Bmp inhibitors Ctgf and Gremlin in islets after 8-wk exposure to high-fat diet. These data imply that Sostdc1 regulates expression of these inhibitors and provide a means by which Sostdc1-null animals show enhanced insulin secretion and glucose homeostasis. Our studies provide insights into Bmp pathway regulation in the endocrine pancreas and reveal new avenues for improving β-cell function under metabolic stress.     

Comprehensive analysis of photoreceptor gene expression and the identification of candidate retinal disease genes.

To identify the full set of genes expressed by mammalian rods, we conducted serial analysis of gene expression (SAGE) by using libraries generated from mature and developing mouse retina. We identified 264 uncharacterized genes that were specific to or highly enriched in rods. Nearly half of all cloned human retinal disease genes are selectively expressed in rod photoreceptors. In silico mapping of the human orthologs of genes identified in our screen revealed that 86 map within intervals containing uncloned retinal disease genes, representing 37 different loci. We expect these data will allow identification of many disease genes, and that this approach may be useful for cloning genes involved in classes of disease where cell type-specific expression of disease genes is observed.     

Rax Homeoprotein Regulates Photoreceptor Cell Maturation and Survival in Association with Crx in the Postnatal Mouse Retina

The Rax homeobox gene plays essential roles in multiple processes of vertebrate retina development. Many vertebrate species possess Rax and Rax2 genes, and different functions have been suggested. In contrast, mice contain a single Rax gene, and its functional roles in late retinal development are still unclear. To clarify mouse Rax function in postnatal photoreceptor development and maintenance, we generated conditional knockout mice in which Rax in maturing or mature photoreceptor cells was inactivated by tamoxifen treatment (Rax iCKO mice). When Rax was inactivated in postnatal Rax iCKO mice, developing photoreceptor cells showed a significant decrease in the level of the expression of rod and cone photoreceptor genes and mature adult photoreceptors exhibited a specific decrease in cone cell numbers. In luciferase assays, we found that Rax and Crx cooperatively transactivate Rhodopsin and cone opsin promoters and that an optimum Rax expression level to transactivate photoreceptor gene expression exists. Furthermore, Rax and Crx colocalized in maturing photoreceptor cells, and their coimmunoprecipitation was observed in cultured cells. Taken together, these results suggest that Rax plays essential roles in the maturation of both cones and rods and in the survival of cones by regulating photoreceptor gene expression with Crx in the postnatal mouse retina.     

Endocardial to Myocardial Notch-Wnt-Bmp Axis Regulates Early Heart Valve Development

Endocardial to mesenchymal transformation (EMT) is a fundamental cellular process required for heart valve formation. Notch, Wnt and Bmp pathways are known to regulate this process. To further address how these pathways coordinate in the process, we specifically disrupted Notch1 or Jagged1 in the endocardium of mouse embryonic hearts and showed that Jagged1-Notch1 signaling in the endocardium is essential for EMT and early valvular cushion formation. qPCR and RNA in situ hybridization assays reveal that endocardial Jagged1-Notch1 signaling regulates Wnt4 expression in the atrioventricular canal (AVC) endocardium and Bmp2 in the AVC myocardium. Whole embryo cultures treated with Wnt4 or Wnt inhibitory factor 1 (Wif1) show that Bmp2 expression in the AVC myocardium is dependent on Wnt activity; Wnt4 also reinstates Bmp2 expression in the AVC myocardium of endocardial Notch1 null embryos. Furthermore, while both Wnt4 and Bmp2 rescue the defective EMT resulting from Notch inhibition, Wnt4 requires Bmp for its action. These results demonstrate that Jagged1-Notch1 signaling in endocardial cells induces the expression of Wnt4, which subsequently acts as a paracrine factor to upregulate Bmp2 expression in the adjacent AVC myocardium to signal EMT.