Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina

The oscillatory expression of Notch signaling in neural progenitors suggests that both repressors and activators of neural fate specification are expressed in the same progenitors. Since Notch1 regulates photoreceptor differentiation and contributes (together with Notch3) to ganglion cell fate specification, we hypothesized that genes encoding photoreceptor and ganglion cell fate activators would be highly expressed in Notch1 receptor-bearing (Notch1⁺) progenitors, directing these cells to differentiate into photoreceptors or into ganglion cells when Notch1 activity is diminished. To identify these genes, we used microarray analysis to study expression profiles of whole retinas and isolated from them Notch1⁺ cells at embryonic day 14 (E14) and postnatal day 0 (P0). To isolate Notch1⁺ cells, we utilized immunomagnetic cell separation. We also used Notch3 knockout (Notch3KO) animals to evaluate the contribution of Notch3 signaling in ganglion cell differentiation. Hierarchical clustering of 6,301 differentially expressed genes showed that Notch1⁺ cells grouped near the same developmental stage retina cluster. At E14, we found higher expression of repressors (Notch1, Hes5) and activators (Dll3, Atoh7, Otx2) of neuronal differentiation in Notch1⁺ cells compared to whole retinal cell populations. At P0, Notch1, Hes5, and Dll1 expression was significantly higher in Notch1⁺ cells than in whole retinas. Otx2 expression was more than thirty times higher than Atoh7 expression in Notch1⁺ cells at P0. We also observed that retinas of wild type animals had only 14% (P < 0.05) more ganglion cells compared to Notch3KO mice. Since this number is relatively small and Notch1 has been shown to contribute to ganglion cell fate specification, we suggested that Notch1 signaling may play a more significant role in RGC development than the Notch3 signaling cascade. Finally, our findings suggest that Notch1⁺ progenitors—since they heavily express both pro-ganglion cell (Atoh7) and pro-photoreceptor cell (Otx2) activators—can differentiate into either ganglion cells or photoreceptors.     

The cadherins fat and dachsous regulate dorsal/ventral signaling in the Drosophila eye

The Drosophila eye is a polarized epithelium in which ommatidia of opposing chirality fall on opposite sides of the eye's midline, the equator. The equator is established in at least two steps: photoreceptors R3 and R4 adopt their fates, and then ommatidia rotate clockwise or counterclockwise in accordance with the identity of these photoreceptors. We report the role of two cadherins, Fat (Ft) and Dachsous (Ds), in conveying the polarizing signal from the D/V midline in the Drosophila eye. In eyes lacking Ft, the midline is abolished. In ft and ds mutant clones, wild-type tissue rescues genetically mutant tissue at the clonal borders, giving rise to ectopic equators. These ectopic equators distort a mosaic analysis of these genes and led to the possible misinterpretation that ft and ds are required to specify the R3 and R4 cell fates, respectively. Our interpretation of these data supports a significantly different model in which ft and ds are not necessarily required for fate determination. Rather, they are involved in long-range signaling during the formation of the equator, as defined by the presence of an organized arrangement of dorsal and ventral chiral ommatidial forms.     

Performance of blue- and green-sensitive photoreceptors of the cricket Gryllus bimaculatus

The compound eye of the cricket Gryllus bimaculatus contains a specialized dorsal rim area (DRA) populated by distinct blue-sensitive photoreceptors responsible for perception of polarized light. The rest of the eye is dominated by green-sensitive photoreceptors. Using patch clamp we studied dissociated ommatidia of nocturnal adults and diurnal eight-instar nymphs with the goals (1) of characterizing the biophysical properties of cricket photoreceptors in general and (2) describing the functionally dissimilar blue- and green-sensitive photoreceptors in terms of voltage-gated channel composition and signal coding. Despite different lifestyles, adult and nymph photoreceptors were indistinguishable. No significant circadian changes were observed in K⁺ currents. In contrast, prominent differences were seen between blue- and green-sensitive photoreceptors. The former were characterized by relatively low absolute sensitivity, high input resistance, slow quantum bumps with long latencies, small light-induced and K⁺ currents and low steady-state depolarization. Information rate, a measure of photoreceptor performance calculated from voltage responses to bandwidth-limited white noise-modulated light contrast, was 87 ± 8 bits s^?1 in green-sensitive photoreceptors vs. 59 ± 14 bits s^?1 in blue-sensitive photoreceptors, implying a limited role of DRA in the perception of visual contrasts. In addition, evidence of electrical coupling between photoreceptors is presented.     

Poly(ADP-ribose) glycohydrolase and poly(ADP-ribose)-interacting protein Hrp38 regulate pattern formation during Drosophila eye development

Drosophila Hrp38, a homolog of human hnRNP A1, has been shown to regulate splicing, but its function can be modified by poly(ADP-ribosyl)ation. Notwithstanding such findings, our understanding of the roles of poly(ADP-ribosyl)ated Hrp38 on development is limited. Here, we have demonstrated that Hrp38 is essential for fly eye development based on a rough-eye phenotype with disorganized ommatidia observed in adult escapers of the hrp38 mutant. We also observed that poly(ADP-ribose) glycohydrolase (Parg) loss-of-function, which caused increased Hrp38 poly(ADP-ribosyl)ation, also resulted in the rough-eye phenotype with disrupted ommatidial lattice and reduced number of photoreceptor cells. In addition, ectopic expression of DE-cadherin, which is required for retinal morphogenesis, fully rescued the rough-eye phenotype of the hrp38 mutant. Similarly, Parg mutant eye clones had decreased expression level of DE-cadherin with orientation defects, which is reminiscent of DE-cadherin mutant eye phenotype. Therefore, our results suggest that Hrp38 poly(ADP-ribosyl)ation controls eye pattern formation via regulation of DE-cadherin expression, a finding which has implications for understanding the pathogenic mechanisms of Hrp38-related Fragile X syndrome and PARP1-related retinal degeneration diseases.     

Abnormalities in cell proliferation and apico-basal cell polarity are separable in Drosophila lgl mutant clones in the developing eye

In homozygous mutants of Drosophila lethal-2-giant larvae (lgl), tissues lose apico-basal cell polarity and exhibit ectopic proliferation. Here, we use clonal analysis in the developing eye to investigate the effect of lgl null mutations in the context of surrounding wild-type tissue. lgl⁻ clones in the larval eye disc exhibit ectopic expression of the G1-S regulator, Cyclin E, and ectopic proliferation, but do not lose apico-basal cell polarity. Decreasing the perdurance of Lgl protein in larval eye disc clones, by forcing extra proliferation of lgl⁻ tissue (using a Minute background), leads to a loss in cell polarity and to more extreme ectopic cell proliferation. Later in development at the pupal stage, lgl mutant photoreceptor cells show aberrant apico-basal cell polarity, but this is not associated with ectopic proliferation, presumably because cells are differentiated. Thus in a clonal context, the ectopic proliferation and cell polarity defects of lgl⁻ mutants are separable. Furthermore, lgl⁻ mosaic eye discs have alterations in the normal patterns of apoptosis: in larval discs some lgl⁻ and wild-type cells at the clonal boundary undergo apoptosis and are excluded from the epithelia, but apoptosis is decreased elsewhere in the disc, and in pupal retinas lgl⁻ tissue shows less apoptosis.     

senseless repression of rough is required for R8 photoreceptor differentiation in the developing Drosophila eye.

An outstanding model to study how neurons differentiate from among a field of equipotent undifferentiated cells is the process of R8 photoreceptor differentiation during Drosophila eye development. We show that in senseless mutant tissue, R8 differentiation fails and the presumptive R8 cell adopts the R2/R5 fate. We identify senseless repression of rough in R8 as an essential mechanism of R8 cell fate determination and demonstrate that misexpression of senseless in non-R8 photoreceptors results in repression of rough and induction of the R8 fate. Surprisingly, there is no loss of ommatidial clusters in senseless mutant tissue and all outer photoreceptor subtypes can be recruited, suggesting that other photoreceptors can substitute for R8 to initiate recruitment and that R8-specific signaling is not required for outer photoreceptor subtype assignment. A genetic model of R8 differentiation is presented.     

A moving wave patterns the cone photoreceptor mosaic array in the zebrafish retina

In this paper, we describe the embryonic origin and patterning of the planar mosaic array of cone photoreceptor spectral subtypes in the zebrafish retina. A discussion of possible molecular mechanisms that might generate the cone mosaic array considers but discards a model that accounts for formation of neuronal mosaics in the inner retina and discusses limitations of mathematical simulations that reproduce the zebrafish cone mosaic pattern. The formation and organization of photoreceptors in the ommatidia of the compound eye of Drosophila is compared with similar features in the developing zebrafish cone mosaic, and a model is proposed that invokes spatiotemporally coordinated cell-cell interactions among cone progenitors to determine the identity and positioning of cone spectral subtypes.     

Retinal degeneration-3 protein attenuates photoreceptor degeneration in transgenic mice expressing dominant mutation of human retinal guanylyl cyclase

Different forms of photoreceptor degeneration cause blindness. Retinal degeneration-3 protein (RD3) deficiency in photoreceptors leads to recessive congenital blindness. We proposed that aberrant activation of the retinal membrane guanylyl cyclase (RetGC) by its calcium-sensor proteins (guanylyl cyclase–activating protein [GCAP]) causes this retinal degeneration and that RD3 protects photoreceptors by preventing such activation. We here present in vivo evidence that RD3 protects photoreceptors by suppressing activation of both RetGC1 and RetGC2 isozymes. We further suggested that insufficient inhibition of RetGC by RD3 could contribute to some dominant forms of retinal degeneration. The R838S substitution in RetGC1 that causes autosomal-dominant cone–rod dystrophy 6, not only impedes deceleration of RetGC1 activity by Ca²⁺GCAPs but also elevates this isozyme''s resistance to inhibition by RD3. We found that RD3 prolongs the survival of photoreceptors in transgenic mice harboring human R838S RetGC1 (R838S⁺). Overexpression of GFP-tagged human RD3 did not improve the calcium sensitivity of cGMP production in R838S⁺ retinas but slowed the progression of retinal blindness and photoreceptor degeneration. Fluorescence of the GFP-tagged RD3 in the retina only partially overlapped with immunofluorescence of RetGC1 or GCAP1, indicating that RD3 separates from the enzyme before the RetGC1:GCAP1 complex is formed in the photoreceptor outer segment. Most importantly, our in vivo results indicate that, in addition to the abnormal Ca²⁺ sensitivity of R838S RetGC1 in the outer segment, the mutated RetGC1 becomes resistant to inhibition by RD3 in a different cellular compartment(s) and suggest that RD3 overexpression could be utilized to reduce the severity of cone–rod dystrophy 6 pathology.     

Clearance of yeast prions by misfolded multi-transmembrane proteins

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) induces the stress response to protect cells against toxicity by the unfolded protein response (UPR), heat shock response (HSR), and ER-associated degradation pathways. Here, we found that over-production of C-terminally truncated multi-transmembrane (MTM) mutant proteins triggers HSR, but not UPR, and clearance of yeast prions [PSI⁺] and [URE3]. One of the mutant MTM proteins, Dip5ΔC-v82, produces a disabled amino-acid permease. Fluorescence microscopy analysis revealed abnormal accumulation of Dip5ΔC-v82 in the ER. Importantly, the mutant defective in the GET pathway, which functions for ER membrane insertion of tail-anchored proteins, failed to translocate Dip5ΔC-v82 to the ER and disabled Dip5ΔC-v82-mediated prion clearance. These findings suggest that the GET pathway plays a pivotal role in quality assurance of MTM proteins, and entraps misfolded MTM proteins into ER compartments, leading to loss-of-prion through a yet undefined mechanism.     

Three distinct roles for notch in Drosophila R7 photoreceptor specification

Receptor tyrosine kinases (RTKs) and Notch (N) proteins are different types of transmembrane receptors that transduce extracellular signals and control cell fate. Here we examine cell fate specification in the Drosophila retina and ask how N acts together with the RTKs Sevenless (Sev) and the EGF receptor (DER) to specify the R7 photoreceptor. The retina is composed of many hundred ommatidia, each of which grows by recruiting surrounding, undifferentiated cells and directing them to particular fates. The R7 photoreceptor derives from a cohort of three cells that are incorporated together following specification of the R2-R5 and R8 photoreceptors. Two cells of the cohort are specified as the R1/6 photoreceptor type by DER activation. These cells then activate N in the third cell (the R7 precursor). By manipulation of N and RTK signaling in diverse combinations we establish three roles for N in specifying the R7 fate. The first role is to impose a block to photoreceptor differentiation; a block that DER activation cannot overcome. The second role, paradoxically, is to negate the first; Notch activation up-regulates Sev expression, enabling the presumptive R7 cell to receive an RTK signal from R8 that can override the block. The third role is to specify the cell as an R7 rather than an R1/6 once RTK signaling has specified the cells as a photoreceptor. We speculate why N acts both to block and to facilitate photoreceptor differentiation, and provide a model for how N and RTK signaling act combinatorially to specify the R1/6 and R7 photoreceptors as well as the surrounding non-neuronal cone cells.     

Septate junctions are required for ommatidial integrity and blood-eye barrier function in Drosophila

The anatomical organization of the Drosophila ommatidia is achieved by specification and contextual placement of photoreceptors, cone and pigment cells. The photoreceptors must be sealed from high ionic concentrations of the hemolymph by a barrier to allow phototransduction. In vertebrates, a blood-retinal barrier (BRB) is established by tight junctions (TJs) present in the retinal pigment epithelium and endothelial membrane of the retinal vessels. In Drosophila ommatidia, the junctional organization and barrier formation is poorly understood. Here we report that septate junctions (SJs), the vertebrate analogs of TJs, are present in the adult ommatidia and are formed between and among the cone and pigment cells. We show that the localization of Neurexin IV (Nrx IV), a SJ-specific protein, coincides with the location of SJs in the cone and pigment cells. Somatic mosaic analysis of nrx IV null mutants shows that loss of Nrx IV leads to defects in ommatidial morphology and integrity. nrx IV hypomorphic allelic combinations generated viable adults with defective SJs and displayed a compromised blood-eye barrier (BEB) function. These findings establish that SJs are essential for ommatidial integrity and in creating a BEB around the ion and light sensitive photoreceptors. Our studies may provide clues towards understanding the vertebrate BEB formation and function.     

Rip3 knockdown rescues photoreceptor cell death in blind pde6c zebrafish

Achromatopsia is a progressive autosomal recessive retinal disease characterized by early loss of cone photoreceptors and later rod photoreceptor loss. In most cases, mutations have been identified in CNGA3, CNGB3, GNAT2, PDE6C or PDE6H genes. Owing to this genetic heterogeneity, mutation-independent therapeutic schemes aimed at preventing cone cell death are very attractive treatment strategies. In pde6c^w59 mutant zebrafish, cone photoreceptors expressed high levels of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3) kinases, key regulators of necroptotic cell death. In contrast, rod photoreceptor cells were alternatively immunopositive for caspase-3 indicating activation of caspase-dependent apoptosis in these cells. Morpholino gene knockdown of rip3 in pde6c^w59 embryos rescued the dying cone photoreceptors by inhibiting the formation of reactive oxygen species and by inhibiting second-order neuron remodelling in the inner retina. In rip3 morphant larvae, visual function was restored in the cones by upregulation of the rod phosphodiesterase genes (pde6a and pde6b), compensating for the lack of cone pde6c suggesting that cones are able to adapt to their local environment. Furthermore, we demonstrated through pharmacological inhibition of RIP1 and RIP3 activity that cone cell death was also delayed. Collectively, these results demonstrate that the underlying mechanism of cone cell death in the pde6c^w59 mutant retina is through necroptosis, whereas rod photoreceptor bystander death occurs through a caspase-dependent mechanism. This suggests that targeting the RIP kinase signalling pathway could be an effective therapeutic intervention in retinal degeneration patients. As bystander cell death is an important feature of many retinal diseases, combinatorial approaches targeting different cell death pathways may evolve as an important general principle in treatment.     

A mosaic analysis of the apterous mutation in Drosophila melanogaster

An analysis of three phenotypes expressed by the apterous-four (ap⁴) mutant of Drosophila melanogaster has been carried out in $\text{ap}{}^4\text{ap}{}^{\mathrm{+}}$ mosaic adults. The wing and haltere deficiency phenotype was found to be autonomous for entirely mutant structures. However, small patches of mutant anterior and posterior wing margin cells can exist in mosaic wings. Examples of duplicated and triplicated lengths of bristle rows were often found associated with the existing mutant patches. About 20% of the mosaics expressed the phenotype of precocious adult death, dying 30–40 hr after eclosion. The focus for this phenotype was located in the posterior region of the abdomen, and a strong correlation was found between expression of this phenotype and the presence of mutant Malpighian tubules. The focus for juvenile hormone deficiency in ap⁴ adults was located near that for precocious adult death; in fact, the two foci may be identical. It is suggested that defective functioning of ap⁴ adult Malpighian tubules results in abnormal hemolymph leading to early death. Inadequate juvenile hormone secretion could result indirectly from impaired glandular functioning attributable to abnormal adult hemolymph.     

Role of kinesin heavy chain in Crumbs localization along the rhabdomere elongation in Drosophila photoreceptor

Background

Crumbs (Crb), a cell polarity gene, has been shown to provide a positional cue for the extension of the apical membrane domain, adherens junction (AJ), and rhabdomere along the growing proximal-distal axis during Drosophila photoreceptor morphogenesis. In developing Drosophila photoreceptors, a stabilized microtubule structure was discovered and its presence was linked to polarity protein localization. It was therefore hypothesized that the microtubules may provide trafficking routes for the polarity proteins during photoreceptor morphogenesis. This study has examined whether Kinesin heavy chain (Khc), a subunit of the microtubule-based motor Kinesin-1, is essential in polarity protein localization in developing photoreceptors.

Methodology/Principal Findings

Because a genetic interaction was found between crb and khc, Crb localization was examined in the developing photoreceptors of khc mutants. khc was dispensable during early eye differentiation and development. However, khc mutant photoreceptors showed a range of abnormalities in the apical membrane domain depending on the position along the proximal-distal axis in pupal photoreceptors. The khc mutant showed a progressive mislocalization in the apical domain along the distal-proximal axis during rhabdomere elongation. The khc mutation also led to a similar progressive defect in the stabilized microtubule structures, strongly suggesting that Khc is essential for microtubule structure and Crb localization during distal to proximal rhabdomere elongation in pupal morphogenesis. This role of Khc in apical domain control was further supported by khc''s gain-of-function phenotype. Khc overexpression in photoreceptors caused disruption of the apical membrane domain and the stabilized microtubules in the developing photoreceptors.

Conclusions/Significance

In summary, we examined the role of khc in the regulation of the apical Crb domain in developing photoreceptors. Since the rhabdomeres in developing pupal eyes grow along the distal-proximal axis, these phenotypes suggest that Khc is essential for the microtubule structures and apical membrane domains during the distal-proximal elongation of photoreceptors, but is dispensable for early eye development.     

Overexpression of Transglutaminase in the Drosophila Wing Imaginal Disc Induced an Extra Wing Crossvein Phenotype

To determine the roles of Drosophila transglutaminase-A (dTG-A), we examined a phenotype induced through ectopic expression of dTG-A. Overexpression of dTG-A in the wing imaginal disc induced an extra wing crossvein phenotype. This phenotype was suppressed by crossing with epidermal growth factor receptor (Egfr) signaling pathway mutant flies. These results indicate that this phenotype, induced by dTG-A, is related to enhancement of the Egfr signaling pathway.