猪Opn4基因cDNA克隆与表达分析

为揭示猪Opn4基因的结构并研究其在猪视网膜中昼夜表达差异与生长各阶段表达规律, 文章克隆了猪Opn4基因全长CDS区序列, 定量检测了Opn4基因在猪视网膜中昼夜的表达量以及其在不同生长阶段的表达量。结果表明, Opn4基因编码区序列为1 437 bp, 编码478个氨基酸, 分子式为C2398H3705N623O651S23; Opn4基因在白昼的表达量极显著高于夜晚(P<0.01); Opn4基因在出生前34 d胎儿视网膜中表达量最低(P<0.01); 1日龄时表达量最高(P<0.01); 1月龄、4月龄、84月龄猪视网膜中Opn4表达量差异不显著(P>0.05)。以上结果提示, Opn4基因可参与调控猪的生物节律, 并且Opn4主要参与出生后猪对外界光照的应答。     

Isolation and characterization of melanopsin (Opn4) from the Australian marsupial Sminthopsis crassicaudata (fat-tailed dunnart)

Melanopsin confers photosensitivity to a subset of retinal ganglion cells and is responsible for many non-image-forming tasks, like the detection of light for circadian entrainment. Recently, two melanopsin genes, Opn4m and Opn4x, were described in non-mammalian vertebrates. However, only one form, Opn4m, has been described in the mammals, although studies to date have been limited to the placentals and have not included the marsupials. We report here the isolation and characterization of an Opn4 gene from an Australian marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata), and present evidence which suggests that the Opn4x gene was lost before the placental/marsupial split. In situ hybridization shows that the expression of Opn4 in the dunnart eye is restricted to a subset of ganglion cells, a pattern previously reported for rodents and primates. These Opn4-positive cells are randomly distributed across the dunnart retina. We also undertook a comparative analysis with the South American marsupial, the grey short-tailed opossum (Monodelphis domestica), and two placental mammals, mouse and human. This approach reveals that the two marsupials show a higher sequence identity than that seen between rodents and primates, despite separating at approximately the same point in time, some 65-85 Myr ago.     

Evolution of Mammalian Opn5 as a Specialized UV-absorbing Pigment by a Single Amino Acid Mutation

Opn5 is one of the recently identified opsin groups that is responsible for nonvisual photoreception in animals. We previously showed that a chicken homolog of mammalian Opn5 (Opn5m) is a G_i-coupled UV sensor having molecular properties typical of bistable pigments. Here we demonstrated that mammalian Opn5m evolved to be a more specialized photosensor by losing one of the characteristics of bistable pigments, direct binding of all-trans-retinal. We first confirmed that Opn5m proteins in zebrafish, Xenopus tropicalis, mouse, and human are also UV-sensitive pigments. Then we found that only mammalian Opn5m proteins lack the ability to directly bind all-trans-retinal. Mutational analysis showed that these characteristics were acquired by a single amino acid replacement at position 168. By comparing the expression patterns of Opn5m between mammals and chicken, we found that, like chicken Opn5m, mammalian Opn5m was localized in the ganglion cell layer and inner nuclear layer of the retina. However, the mouse and primate (common marmoset) opsins were distributed not in the posterior hypothalamus (including the region along the third ventricle) where chicken Opn5m is localized, but in the preoptic hypothalamus. Interestingly, RPE65, an essential enzyme for forming 11-cis-retinal in the visual cycle is expressed near the preoptic hypothalamus of the mouse and common marmoset brain but not near the region of the chicken brain where chicken Opn5m is expressed. Therefore, mammalian Opn5m may work exclusively as a short wavelength sensor in the brain as well as in the retina with the assistance of an 11-cis-retinal-supplying system.     

Activation of the blue opsin gene in cone photoreceptor development by retinoid-related orphan receptor beta

Color vision requires the expression of opsin photopigments with different wavelength sensitivities in retinal cone photoreceptors. The basic color visual system of mammals is dichromatic, involving differential expression in the cone population of two opsins with sensitivity to short (S, blue) or medium (M, green) wavelengths. However, little is known of the factors that directly activate these opsin genes and thereby contribute to the S or M opsin identity of the cone. We report that the orphan nuclear receptor RORbeta (retinoid-related orphan receptor beta) activates the S opsin gene (Opn1sw) through binding sites upstream of the gene. RORbeta lacks a known physiological ligand and activates the Opn1sw promoter modestly alone but strongly in synergy with the retinal cone-rod homeobox factor (CRX), suggesting a cooperative means of enhancing RORbeta activity. Comparison of wild-type and mutant lacZ reporter transgenes showed that the RORbeta-binding sites in Opn1sw are required for expression in mouse retina. RORbeta-deficient mice fail to induce S opsin appropriately during postnatal cone development. Photoreceptors in these mice also lack outer segments, indicating additional functions for RORbeta in photoreceptor morphological maturation. The results identify Opn1sw as a target gene for RORbeta and suggest a key role for RORbeta in regulating opsin expression in the color visual system.     

Characterization of mouse orthologue of ELOVL4: genomic organization and spatial and temporal expression

Mutations in ELOVL4 are associated with dominant macular degeneration (adMD/STGD3). This gene is highly expressed in the retina and is conserved through evolution. Here we report the genomic organization of the mouse orthologue of ELOVL4 and its temporal and spatial expression. A significant amount of ELOVL4 mRNA expression is detected in the adult retina, brain, skin, testis, and lens. During development, expression is first noted at embryonic day 7 (E7). A significant level of the mRNA is observed both in brain and in eyes at postnatal day 1 (P1), after which levels decrease in the brain and increase in the retina until they stabilize at P30. ELOVL4 protein is evident in the ocular tissues by E10.5 and becomes restricted predominantly to the photoreceptor layer in the mature retina. These observations suggest that ELOVL4 may play an important role in embryonic development and in maintaining normal physiology of retina and brain at later stages of development.     

Expression of connexins 36, 43, and 45 during postnatal development of the mouse retina

Gap junction channels formed by connexins (Cx) may play essential roles in some processes that occur during retinal development, such as apoptosis and calcium wave spread. The present study was undertaken to determine the distribution pattern of Cx36, Cx43, and Cx45 by immunofluorescence, as well as their gene expression levels by quantitative PCR during postnatal development of the mouse retina. Our results showed an increased expression of neuronal Cx36 from P1 until P10, when this Cx reached adult levels, and it was mainly distributed in the outer and inner plexiform layers. In turn, Cx43 was almost absent in retinal progenitor cells at P1, it became more prominent in glial cell processes about P10, and did not change until adulthood. Double-labeling studies in situ and in vitro with antivimentin, a Müller cell marker, confirmed that Cx43 was expressed by these cells. In addition, quantitative PCR showed that Cx43 and vimentin shared very similar temporal expression patterns. Finally, in contrast to Cx36 and Cx43, Cx45 mRNA was strongly down-regulated during development. In early postnatal days, Cx45 was seen ubiquitously distributed throughout the retina in cells undergoing proliferation and differentiation, as well in differentiated neurons. In adult retina, this protein had a more restricted distribution both in neurons and glial cells, as confirmed in situ and in vitro. In conclusion, we observed a distinct temporal expression pattern for Cx36, Cx43, and Cx45, which is probably related to particular roles in retinal function and maintenance of homeostasis during development of the mouse retina.     

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.     

Postnatal myosin heavy chain isoform expression in normal mice and mice null for IIb or IId myosin heavy chains

The patterns of myosin heavy chain (MyHC) isoform expression in the embryo and in the adult mouse are reasonably well characterized and quite distinct. However, little is known about the transition between these two states, which involves major decreases and increases in the expression of several MyHC genes. In the present study, the expression of seven sarcomeric MyHCs was analyzed in the hindlimb muscles of wild-type mice and in mice null for the MyHC IIb or IId/x genes at several time points from 1 day of postnatal life (dpn) to 20 dpn. In early postnatal life, the developmental isoforms (embryonic and perinatal) comprise >90% of the total MyHC expression, while three adult fast isoforms (IIa, IIb, and IId) comprise <1% of the total MyHC protein. However, between 5 and 20 dpn their expression increases to comprise >90% of the total MyHC. Expression of each of the three adult fast isoforms occurs in a spatially and temporally distinct manner. We also show that alpha MyHC, which is almost exclusively expressed in the heart, is expressed in scattered fibers in all hindlimb muscles during postnatal development. Surprisingly, the timing and localization of expression of the MyHC isoforms is unchanged in IIb and IId/x null mice, although the magnitude of expression is altered for some isoforms. Together these data provide a comprehensive overview of the postnatal expression pattern of the sarcomeric MyHC isoforms in the mouse hindlimb.     

The expression analysis of Sfrs10 and Celf4 during mouse retinal development

Processing of mRNAs including, alternative splicing (AS), mRNA transport and translation regulation are crucial to eukaryotic gene expression. For example, >90% of the genes in the human genome are known to undergo alternative splicing thereby expanding the proteome production capacity of a limited number of genes. Similarly, mRNA export and translation regulation plays a vital role in regulating protein production. Thus, it is important to understand how these RNA binding proteins including alternative splicing factors (ASFs) and mRNA transport and translation factors regulate these processes. Here we report the expression of an ASF, serine-arginine rich splicing factor 10 (Sfrs10) and a mRNA translation regulation factor, CUGBP, elav like family member 4 (Celf4) in the developing mouse retina. Sfrs10 was expressed throughout postnatal (P) retinal development and was observed progressively in newly differentiating neurons. Immunofluorescence (IF) showed Sfrs10 in retinal ganglion cells (RGCs) at P0, followed by amacrine and bipolar cells, and at P8 it was enriched in red/green cone photoreceptor cells. By P22, Sfrs10 was observed in rod photoreceptors in a peri-nuclear pattern. Like Sfrs10, Celf4 expression was also observed in the developing retina, but with two distinct retinal isoforms. In situ hybridization (ISH) showed progressive expression of Celf4 in differentiating neurons, which was confirmed by IF that showed a dynamic shift in Celf4 localization. Early in development Celf4 expression was restricted to the nuclei of newly differentiating RGCs and later (E16 onwards) it was observed in the initial segments of RGC axons. Later, during postnatal development, Celf4 was observed in amacrine and bipolar cells, but here it was predominantly cytoplasmic and enriched in the two synaptic layers. Specifically, at P14, Celf4 was observed in the synaptic boutons of rod bipolar cells marked by Pkc-α. Thus, Celf4 might be regulating AS early in development besides its known role of regulating mRNA localization/translation. In all, our data suggests an important role for AS and mRNA localization/translation in retinal neuron differentiation.     

Development of the mouse retina: emerging morphological diversity of the ganglion cells

The time course and regulatory mechanisms of dendritic development are subjects of intense interest. We approached these problems by investigating dendritic morphology of retinal ganglion cells (RGCs) at four early postnatal stages. The RGCs develop from a diffusely stratified and poorly differentiated group at birth (P0), to 16 distinct, morphologically well-defined subtypes before eye opening (P13). Even before bipolar cells make synaptic contacts with the RGCs (P8), most adultlike RGC subtypes are already present. Similar to previous studies in other mammalian species, our results indicate that the initiation of the RGC morphological maturation is independent of light stimulation and of formation of glutamatergic synapses. This study narrowed down the window of RGCs morphological maturation and highlighted a few early postnatal events as potential factors controlling the developmental process. Because mouse is the most popular mammalian model for genetic manipulation, this study provided a foundation for further exploring regulatory mechanisms of RGC dendritic development.     

Structure of GABAB receptors in rat retina

In the search for yet unknown subtypes of GABAB receptors, the subunit architecture of GABAB receptors in the retina was analyzed using selective antisera. Immunopurification of the splice variants GABAB1a and GABAB1b demonstrated that both were associated with GABAB2. Quantitative immunoprecipitation experiments indicated that practical the entire GABAB receptor population in the retina consists of the receptor subtypes GABAB1a/GABAB2 and GABAB1b/GABAB2, although low levels of GABAB1c/GABAB2 cannot be excluded. The data rule out the existence of GABAB receptors containing the splice variants GABAB1d and GABAB1e. Moreover, no evidence for homomeric GABAB1 receptors was found. Among the splice variants, GABAB1a is by far the predominant one in neonatal and adult retina, whereas GABAB1b is expressed only late in postnatal development and in the adult retina. Since GABAB1a is expressed at high levels before functional synapses are formed, this specific receptor subtype might be involved in the maturation of the retina. Finally, subcellular fractionation demonstrated that GABAB1a, but not GABAB1b, is present in postsynaptic densities, suggesting a differential pre- and postsynaptic localisation of both splice variants.     

Localization of the ultraviolet-sensor Opn5m and its effect on myopia-related gene expression in the late-embryonic chick eye

Recent studies show that exposure to ultraviolet (UV) light suppresses ocular elongation, which causes myopia development. However, the specific mechanisms of this process have not been elucidated. A UV-sensor, Opsin 5 (Opn5) mRNA was shown to be present in extraretinal tissues. To test the possibility that UV-signals mediated by Opn5 would have a direct effect on the outer connective tissues of the eye, we first examined the expression patterns of a mammalian type Opn5 (Opn5m) in the late-embryonic chicken eye. Quantitative PCR showed Opn5m mRNA expression in the cornea and sclera. The anti-Opn5m antibody stained a small subset of cells in the corneal stroma and fibrous sclera. We next assessed the effect of UV-A (375 nm) irradiation on the chicken fibroblast cell line DF-1 overexpressing chicken Opn5m. UV-A irradiation for 30 min significantly increased the expression of Early growth response 1 (Egr1), known as an immediate early responsive gene, and of Matrix metalloproteinase 2 (Mmp2) in the presence of retinal chromophore 11-cis-retinal. In contrast, expression of Transforming growth factor beta 2 and Tissue inhibitor of metalloproteinase 2 was not significantly altered. These results indicate that UV-A absorption by Opn5m can upregulate the expression levels of Egr1 and Mmp2 in non-neuronal, fibroblasts. Taken together with the presence of Opn5m in the cornea and sclera, it is suggested that UV-A signaling mediated by Opn5 in the extraretinal ocular tissues could influence directly the outer connective tissues of the chicken late-embryonic eye.     

Developmental changes in lysophospholipid receptor expression in rodent heart from near-term fetus to adult

Lysophospholipids (LPs) are small signaling lipids that regulate diverse physiological and pathological processes through G protein-coupled receptors. To investigate the function of LP signaling in heart organogenesis and maturation, we measured the expression of 10 confirmed LP receptors (Lpar1-5 and S1pr1-5) in rat heart from embryonic day 19.5 (E19.5d) to postnatal week 12 (P12w). The expression of Lpar3 mRNA peaked at 37-fold higher than adult expression at P1d, while the expression levels of Lpar1 and Lpar4 increased markedly after P1d and peaked at 19- and 48-folds of adult expression on P7d. The expression levels of all three receptor mRNAs were significantly reduced by P21d and remained low thereafter. Expression of the corresponding receptor proteins also peaked during the early postnatal period but the subsequent decline was less dramatic from P14d to P12w compared to mRNA expression. In contrast, S1pr1 and S1pr3 exhibited more gradual developmental changes. Although early expression was higher than mature expression (3- to 6-fold), these receptors were still strongly expressed at P12w. The other isotypes examined, Lpar2, Lpar5, S1pr4, and S1pr5, were very weakly expressed at all developmental stages. Analysis of receptor distribution within the developing heart (P1d) revealed that Lpar1, Lpar3, and Lpar4 were expressed in the myocardium of all four chambers but not in valves, while Lpar3 was also uniquely expressed in the aorta and coronary vessels. Western blots revealed that the developmental changes in Lpar1, Lpar3, and Lpar4 protein expression mirrored changes in β-actin and β-tubulin expression. The increase in Lpar1 and Lpar4 receptors from P1d to P7d corresponds to the period of rapid myocardial growth and functional maturation. Moreover, the relatively high expression of Lpar1, Lpar3, and Lpar4 in the late prenatal rat heart suggests that these LPA receptors may also contribute to organogenesis. The increase in Lpar3 and Lpar4 expression concomitant with rising expression of cytoskeleton proteins further suggests a possible role for LPA signaling in cytoskeletal remodeling during cardiac development.