The expression and function of midkine in the vertebrate retina.

UNLABELLED: The functional role of midkine during development, following injury and in disease has been studied in a variety of tissues. In this review, we summarize what is known about midkine in the vertebrate retina, focusing largely on recent studies utilizing the zebrafish (Danio rerio) as an animal model. Zebrafish are a valuable animal model for studying the retina, due to its very rapid development and amazing ability for functional neuronal regeneration following neuronal cell death. The zebrafish genome harbours two midkine paralogues, midkine-a (mdka) and midkine-b (mdkb), which, during development, are expressed in nested patterns among different cell types. mdka is expressed in the retinal progenitors and mdkb is expressed in newly post-mitotic cells. Interestingly, studies of loss- and gain-of-function in zebrafish larvae indicate that midkine-a regulates cell cycle kinetics. Moreover, both mdka and mdkb are expressed in different cell types in the normal adult zebrafish retina, but after light-induced death of photoreceptors, both are up-regulated and expressed in proliferating Müller glia and photoreceptor progenitors, suggesting an important and (perhaps) coincident role for these cytokines during stem cell-based neuronal regeneration. Based on its known role in other tissues and the expression and function of the midkine paralogues in the zebrafish retina, we propose that midkine has an important functional role both during development and regeneration in the retina. Further studies are needed to understand this role and the mechanisms that underlie it. LINKED ARTICLES: This article is part of a themed section on Midkine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-4.

NeuroD regulates proliferation of photoreceptor progenitors in the retina of the zebrafish.

neuroD is a member of the family of proneural genes, which function to regulate the cell cycle, cell fate determination and cellular differentiation. In the retinas of larval and adult teleosts, neuroD is expressed in two populations of post-mitotic cells, a subset of amacrine cells and nascent cone photoreceptors, and proliferating cells in the lineages that give rise exclusively to rod and cone photoreceptors. Based on previous studies of NeuroD function in vitro and the cellular pattern of neuroD expression in the zebrafish retina, we hypothesized that within the mitotic photoreceptor lineages NeuroD selectively regulates aspects of the cell cycle. To test this hypothesis, gain and loss-of-function approaches were employed, relying on the inducible expression of a NeuroD(EGFP) fusion protein and morpholino oligonucleotides to inhibit protein translation, respectively. Conditional expression of neuroD causes cells to withdraw from the cell cycle, upregulate the expression of the cell cycle inhibitors, p27 and p57, and downregulate the cell cycle progression factors, Cyclin B1, Cyclin D1, and Cyclin E2. In the absence of NeuroD, cells specific for the rod and cone photoreceptor lineage fail to exit the cell cycle, and the number of cells expressing Cyclin D1 is increased. When expression is ectopically induced in multipotent progenitors, neuroD promotes the genesis of rod photoreceptors and inhibits the genesis of Müller glia. These data show that in the teleost retina NeuroD plays a fundamental role in photoreceptor genesis by regulating mechanisms that promote rod and cone progenitors to withdraw from the cell cycle. This is the first in vivo demonstration in the retina of cell cycle regulation by NeuroD.

Controlling diastereoselectivity in the reactions of enantiomerically pure alpha-bromoacyl-imidazolidinones with nitrogen nucleophiles: substitution reactions with retention or inversion of configuration.

Diastereoselective substitution reactions of [small alpha]-bromoacyl-imidazolidinones with nitrogen nucleophiles can be promoted with either retention or inversion of configuration by carrying out reactions under epimerising or non-epimerising conditions.

Interferometer-controlled scanning transmission X-ray microscopes at the Advanced Light Source.

Two new soft X-ray scanning transmission microscopes located at the Advanced Light Source (ALS) have been designed, built and commissioned. Interferometer control implemented in both microscopes allows the precise measurement of the transverse position of the zone plate relative to the sample. Long-term positional stability and compensation for transverse displacement during translations of the zone plate have been achieved. The interferometer also provides low-distortion orthogonal x, y imaging. Two different control systems have been developed: a digital control system using standard VXI components at beamline 7.0, and a custom feedback system based on PC AT boards at beamline 5.3.2. Both microscopes are diffraction limited with the resolution set by the quality of the zone plates. Periodic features with 30 nm half period can be resolved with a zone plate that has a 40 nm outermost zone width. One microscope is operating at an undulator beamline (7.0), while the other is operating at a novel dedicated bending-magnet beamline (5.3.2), which is designed specifically to illuminate the microscope. The undulator beamline provides count rates of the order of tens of MHz at high-energy resolution with photon energies of up to about 1000 eV. Although the brightness of a bending-magnet source is about four orders of magnitude smaller than that of an undulator source, photon statistics limited operation with intensities in excess of 3 MHz has been achieved at high energy resolution and high spatial resolution. The design and performance of these microscopes are described.

Persistent neurogenesis in the teleost retina: evidence for regulation by the growth-hormone/insulin-like growth factor-I axis.

Based on results from previous studies (J. Comp. Neurol. 394 (1998) 386, 395), it was hypothesized that the persistent neurogenesis in the retina of teleost fish is modulated by insulin-like growth factor-I (IGF-I), which, in turn, is regulated by growth hormone (GH). Two approaches were undertaken to test this hypothesis. First, a variety of techniques were used to determine if IGF-I and the IGF-I receptor (IGF-IR) are expressed in the retina. Second, GH was injected into animals to stimulate IGF-I synthesis in target tissues, and IGF-I expression and cell proliferation in the retina was quantitatively assayed. Reverse transcriptase-polymerase chain reaction, screening a retinal cDNA library and Northern analysis showed that genes encoding IGF-I and IGF-IR are expressed in the retina of goldfish. In situ hybridization showed that IGF-IR is expressed by retinal progenitors and all differentiated retinal neurons. Intraperitoneal injections of GH elevate IGF-I mRNA levels in the liver, brain and retina and produce a dose-dependent change in the proliferation of stem cells and progenitors in the retina. These data indicate that the principal components of the IGF-I signaling cascade are present in the retinas of teleosts, and we suggest these elements mediate the persistent, growth-associated neurogenesis in this tissue.

Expression of the insulin receptor in the retina of the goldfish.

PURPOSE: Insulin is a peptide growth factor that is active in most tissues, both during development and in adulthood. The action of insulin is through its specific membrane receptor. Previously retinal progenitors in the adult goldfish were shown to proliferate vigorously when exposed to insulin in vitro.(1) The present study was undertaken to clone and characterize partial cDNAs that encode the goldfish's insulin receptor (IR) and to establish the cellular pattern of expression of this gene in the retina. METHODS: Standard methods were used for RNA isolation, reverse transcription-polymerase chain reaction, Northern blot analysis, and in situ hybridization. RESULTS: Multiple clones were isolated that, based on sequence analysis, segregated into two groups, presumed to represent two genes that encode the IR. These clones were designated goldfish IR-1 (gfIR-1) and goldfish IR-2 (gfIR-2). Northern blot analysis showed that both genes are expressed in multiple tissues, including the retina. Both gfIR-1 and -2 give rise to a single, major transcript, but the sizes of the two transcripts are different. In situ hybridizations using digoxygenin-labeled riboprobes showed that gfIR-1 and -2 are expressed by all differentiated retinal neurons as well as neuronal progenitors in the circumferential germinal zone. CONCLUSIONS: These data demonstrate that the IR is expressed in the retina of the goldfish, and, on the basis of the cellular pattern of expression, suggest that insulin may act both to regulate neurogenesis and influence the function of differentiated neurons. The cellular coexpression of the receptors for both insulin-like growth factor (IGF) 1 and insulin suggests that neurons and/or neuronal progenitors in the retina of the goldfish may contain hybrid IGF-1/insulin receptors.

Novel rearrangements of sesquiterpenoid panasinsane derivatives under acidic conditions.

The sesquiterpenoid panasinsane derivatives 11 and 14-16 have been prepared from caryophyllene oxide (7). The novel rearrangement reactions of compounds 11 and 14 under TCNE-catalyzed solvolysis conditions and the reactions of compounds 15 and 16 under superacid conditions (HSO3F/Et2O, -63 degrees C) have been investigated. The ginsenol derivative 17 is obtained from compounds 11 and 14 under TCNE-catalyzed conditions. The rearrangement of compounds 15 and 16 under superacid conditions leads to the novel sesquiterpene derivatives (1S,4S,7S,10S,11S)-3,3,10,11-tetramethyltricyclo[5.3.1.0(4,10)]undecan-1,11-yl sulfate (19) and (1S,4S,5S,8S)-2,2,4,8-tetramethyl tricyclo[3.3.2.1(4,8)]undecan-11-one (20). The influence of the secondary hydroxyl group at C-5 of the panasinsane derivatives on the course of these rearrangements is discussed.

Putative stem cells and the lineage of rod photoreceptors in the mature retina of the goldfish.

The retinas of teleost fish grow continuously, in part, by neuronal hyperplasia and when lesioned will regenerate. Within the differentiated retina, the growth-associated hyperplasia results in the generation of new rod photoreceptors only, whereas injury-induced neurogenesis results in the regeneration of all retinal cell types. It is believed, however, that both new rod photoreceptors and regenerated neurons originate from the same populations of intrinsic progenitors. Experiments are described here that attempt to identify in the normal retina of goldfish neuronal progenitors intrinsic to the retina, particularly those which have remained cryptic because they divide infrequently. Long-term, systemic exposure to bromodeoxyuridine (BrdU) was used to label these cells. Five populations of proliferative cells were labeled: microglia, which are briefly described but not studied further; retinal progenitors in the circumferential germinal zone (CGZ); and rod precursors in the outer nuclear layer (ONL), both of which have been well characterized previously; and two populations of slowly-dividing cells in the inner nuclear layer (INL). The majority of these cells have a fusiform morphology, whereas the remaining ones are spherical. Longitudinal BrdU labeling suggests that the fusiform cells migrate to the ONL to replenish the pool of rod precursors. A subset of the spherical cells express pax6, although none are stained with markers of differentiated amacrine or bipolar cells. It is hypothesized that these rare, pax6-expressing cells are retinal stem cells, which give rise to the pax6-negative fusiform cells. Based on these data, two models are proposed: the first describes the lineage of rod photoreceptors in goldfish; the second is a consensus model of neurogenesis in the retinas of all teleosts.

The Remarkable Stable Emerald Green C60 F15 [CBr(CO2 Et)2 ]3 : The First [60]Fullerene That Is also the First [18]Trannulene.

It is both an [18]trannulene and a [60]fullerene. It is aromatic and a hexa-substituted benzene. It is formed by the first proven example of an SN 2' reaction in a fullerene. It is intensely colored and stable. It is C60 F15 [CBr(CO2 Et)2 ]3 , the first example of a new class of fullerene derivatives (see Schlegel diagram).

Cerium masquerading as a group 4 element: synthesis, structure and computational characterisation of [CeCl(N(SiMe3)2)3].

Oxidation of the three-coordinate cerium amide [Ce(N-(SiMe3)2)3] with TeCl4 in toluene solution yields purple, diamagnetic [CeCl(N(SiMe3)2)3], whose structure has been examined by X-ray crystallographic and computational methods.

Preventing HIV infection: lessons from Mwanza and Rakai.

PIP: The impact of enhanced syndromic diagnosis of symptomatic sexually transmitted infections (STIs) upon the incidence of HIV infections was evaluated in 8 paired villages in Mwanza, Tanzania, over a 2-year period. Shortly thereafter, a study was conducted in Uganda's Rakai district which focused upon treating all members of 5 clusters of paired communities, including those with symptomatic and asymptomatic STIs. In August 1995, the results of the Mwanza study showed that almost 40% of HIV infections had been prevented in the communities receiving the intervention. No other HIV intervention has had such a major effect upon infection rates. In contrast, however, no HIV infections were prevented in the Rakai intervention communities. The Mwanza results could reflect the short-term impact of STD prevention and control in an immature epidemic, while the Rakai study reflects the short-term impact in a mature epidemic. The probability of transmission, the duration of infectiousness, and the number of sex partners are discussed as factors which influence the generation of an HIV epidemic in a susceptible population. The 2 studies' results indicate that STD prevention and control is feasible, effective, and affordable.^ieng

Insulitis and mechanisms of disease resistance: studies in an animal model of insulin dependent diabetes mellitus.

Insulin dependent diabetes mellitus (IDDM) is an autoimmune disease characterised by extreme insulin deficiency due to an overall decrease in the mass of properly functioning beta-cells. This reduction occurs as a result of insulitis. the outcome of which will depend upon the intensity of the cytotoxic attack and the ability of beta-cells to resist and repair immune mediated cell damage. To further elucidate the relationship between the insulitis process and beta-cell defence and repair mechanisms in the prevention of diabetes we have studied a unique subgroup of diabetes prone (DP) BB/S rats which have demonstrated an ability to recover from IDDM (BB/S-R). Animals were diagnosed as diabetic at 115 days of age, subsequently receiving insulin therapy (1.49+/-0.1 IU/day) for a total of 19.7 days during 1 to 4 episodes of IDDM. Following a prolonged symptom-free period of 90 days, an IPGTT revealed that BB/S-R rats possessed normal glycaemic control. Islets were isolated from the BB/S-R rats and their glucose-stimulated insulin response was shown to be comparable to Wistar control islets. Furthermore, control and BB/S-R islets showed both a similar structural integrity and insulin content. BB/S-R islets cultured for 24 hr in IL-1beta (10(-13) M) maintained a significant insulin secretory response to glucose in contrast to Wistar controls in which the response was completely inhibited. Nitrite production was induced by IL-1beta, in a dose-dependent manner, in control islets whereas there was no significant increase in production in the islets of BB/S-R rats. These findings suggest that previous immune directed beta-cell attack may induce a state of increased resistance to subsequent deleterious effects of cytokine-mediated cytotoxicity. Overall therefore, the present study shows how the "recovered" BB/S-R rat model provides a unique opportunity to assess the direct effects of insulitis on pancreatic islets and how this interaction may subsequently determine disease outcome.

Thermally Stable Heterobinuclear Bivalent Group 14 Metal Complexes Ar2 M-Sn[1,8-(NR)2 C10 H6 ] (M=Ge, Sn; Ar=2,6-(Me2 N)2 C6 H3 ; R=CH2 tBu).

The first thermally robust Ge(II) -Sn(II) compound 1 and the structurally characterized Sn(II) -Sn(II) analogue 2, which maintain their structural integrity in solution, were obtained by treating MAr2 (M=Ge, Sn; Ar=2,6-(Me2 N)2 C6 H3 ) with Sn[1,8-(NR2 )2 C10 H6 ] (R=CH2 tBu). On the basis of structural and spectroscopic data, the M-Sn bond is regarded as the interaction of a MAr2 donor with an Sn[1,8-(NR2 )2 C10 H6 ] acceptor.

Calcium channel beta 4 (CACNB4): human ortholog of the mouse epilepsy gene lethargic.

The mouse neurological mutant lethargic (lh) is characterized by ataxia, focal myoclonus, and absence epilepsy due to a loss-of-function mutation in the beta4 subunit of the voltage-gated calcium channel. To evaluate the role of this channel subunit in human neurological disease, we determined the chromosomal location and intron/exon structure of the human CACNB4 gene. The 1560-bp open reading frame of the CACNB4 cDNA predicts a 58-kDa protein with an amino acid sequence that is 99% identical to the rat protein. The 13 coding exons of CACNB4 span >55 kb of genomic DNA. Human cerebellar RNA contains one major CACNB4 transcript that is 9 kb in length. Expression of CACNB4 was detected in cerebellum, kidney, testis, retina, lymphoblasts, and circulating lymphocytes. Retinal transcripts were localized by in situ hybridization to ganglion cells and the inner nuclear layer. Analysis of the GeneBridge 4 radiation hybrid mapping panel localized CACNB4 to position 791 cR on human chromosome 2, in a conserved linkage group on human 2q22-q31 and mouse chromosome 2. We localized CACNB4 to the 1.3-Mb YAC clone 952F10 in Whitehead contig WC861, along with the polymorphic markers D2S2236 and D2S2299. The chromosomal linkage of three of the four beta subunit genes to homeobox gene clusters associates the evolutionary origin of the beta gene family with the events that generated the four HOX clusters early in vertebrate evolution.

Insulin-related growth factors stimulate proliferation of retinal progenitors in the goldfish.

The retina of the adult goldfish grows throughout the life of the animal, in part, by the continual addition of new neurons. Further, destruction of extant neurons in this tissue stimulates neuronal regeneration. In an attempt to identify growth factors that regulate both normal and injury-stimulated neurogenesis, we used organ culture techniques and tested nine peptide growth factors for their ability to modulate cell proliferation in both normal retinas and retinas with lesions. Of the growth factors tested, only the insulin-related peptides (insulin and insulin-like growth factors I and II) consistently stimulated proliferation, and this was restricted to the retinal progenitors within the circumferential germinal zone. None of the growth factors tested stimulated proliferation of rod precursors (cells in the mature retina whose progeny are exclusively rod photoreceptors) or the injury-stimulated retinal progenitors. Although the negative data are subject to multiple interpretations, these data suggest that in the retina of the adult goldfish, insulin-related peptides regulate proliferation of retinal progenitors within the circumferential germinal zone, but molecules that modulate the proliferation of the rod precursors or injury-induced retinal progenitors in the retina of the adult goldfish have yet to be identified.

Insulin-like growth factor-I binds in the inner plexiform layer and circumferential germinal zone in the retina of the goldfish.

Results of the previous study suggest that insulin-related peptides regulate proliferation of retinal progenitors in the adult goldfish. Because of their known roles in retinal neurogenesis, we have chosen to focus future studies on insulin-like growth factor I (IGF-I) and the IGF-I receptor. In the study described here, we characterized the spatial distribution and specificity of IGF-I binding sites in the retina of the adult goldfish by performing receptor-binding autoradiography with [125I]-IGF-I alone and with unlabeled IGF-I-related molecules (IGF-I, IGF-II, insulin, and des-[1-3]-IGF-I) as competitive inhibitors of [125I]-IGF-I binding. The results of these experiments show that IGF-I binds in two locations in the retina of the adult goldfish, within the inner plexiform layer of the differentiated retina and the circumferential germinal zone. The competition experiments suggest that [125I]-IGF-I binds at sites specific for IGF-I, and that both IGF-I receptors and IGF-I binding proteins are present in the retina.

Pax2 expression and retinal morphogenesis in the normal and Krd mouse.

The Kidney and retinal defects (Krd) mouse carries a 7-cM transgene-induced deletion on chromosome 19 that includes the Pax2 locus. Adult mice heterozygous for the Krd deletion (Krd/+) are haploid for Pax2 and have a variable, semidominant phenotype characterized by structural defects of the kidney, retina, and optic disc. Renal and ocular anomalies present in heterozygous Pax2 mutants in both mice and humans support the hypothesis that haploinsufficiency of Pax2 underlies the Krd phenotype. To understand the embryonic basis of ocular defects observed in adult Krd/+ mice, we used immunohistochemistry, digital three-dimensional reconstructions, and quantitative morphometry to examine Pax2 protein distribution and ocular development in normal and Krd/+ mice from E10.5 to P2. In +/+ embryos, Pax2 immunopositive (Pax2+) cells demarcate the embryonic fissure as it forms in the ventral optic cup and optic stalk. After closure of the embryonic fissure, Pax2 immunostaining disappears from the ventral retina, but persists in a cuff of cells encircling the developing optic disc, the site where ganglion cell axons exit the retina. In Krd/+ embryos, Pax2+ cells in the posterior optic cup and the optic stalk undergo abnormal morphogenetic movements and the embryonic fissure fails to form normally. This results in an abnormal organization of the Pax2+ cells and ganglion cell axons at the nascent optic disc. The abnormal morphogenetic movements of the Pax2+ cells in the embryonic retina and optic stalk and the initial misrouting of the ganglion cell axons give rise to retinal and optic disc defects observed in the adult Krd/+ mice.

Vsx-1 and Vsx-2: two Chx10-like homeobox genes expressed in overlapping domains in the adult goldfish retina.

The genetic linkages of the murine ocular retardation mutation with the Chx10 gene and the murine small eye mutation with the Pax-6 gene has demonstrated the importance of Paired class homeobox genes in the development of the mammalian retina. Previously, we identified a Paired-class homeobox gene, Vsx-1, whose expression in the adult goldfish retina is restricted to the inner nuclear layer (INL) and to postmitotic, differentiating progenitor cells in the growth zone at the retinal peripheral margin, where neurogenesis continues throughout life. Here, we report the molecular cloning and expression pattern of a new Paired class homeobox gene, Vsx-2, in the adult goldfish retina. Like Vsx-1, Vsx-2 expression is highly restricted to the retina in the adult goldfish and overlaps with Vsx-1 expression in the mature INL. At the peripheral margin, Vsx-2 is expressed in mitotically active neuronal progenitors and is downregulated as these cells become postmitotic and begin to differentiate. Comparison of the amino acid sequences of Vsx-2, Vsx-1, Chx10, and C. elegans ceh-10 reveal a conserved homeodomain and a unique domain termed the CVC domain. The similarities of the Vsx-2, Vsx-1, and Chx10 expression patterns suggest that genes containing the CVC domain have conserved functions during retinal development in vertebrates.