Suppression of lens growth by alphaA-crystallin promoter-driven expression of diphtheria toxin results in disruption of retinal cell organization in zebrafish

In order to study lens-retina relationships during development, we cloned the zebrafish alphaA-crystallin cDNA and its promoter region. Using a 2.8-kb fragment of the zebrafish alphaA-crystallin promoter (z(alpha)Acry), we expressed the diphtheria toxin A fragment (DTA) in zebrafish embryos in a lens-specific manner. Injection of the z(alpha)Acry-DTA plasmid into eggs at the one-or two-cell stage resulted in the formation of small eyes, in which both lens and retina were reduced in size. In the DTA-expressing lenses, their fiber structure was disorganized, indicating that normal lens development had been abrogated. The neural retina also showed abnormal development, although this tissue did not express DTA. Lamination in the retina did not develop well, and molecular markers for the outer and inner plexiform layers were either abnormally expressed or absent. However, cell type-specific markers of ganglion and bipolar cells, as well as photoreceptors, were expressed in appropriate positions, indicating that initial differentiation of these retinal subpopulations occurred in the DTA-expressing embryos. Cell proliferation also proceeded normally in these embryos, although apoptosis was enhanced. These results suggest that the differentiated lens plays a critical role in the morphogenetic organization of retinal cells during eye development in zebrafish embryos.     

The possibility of lens formation in explants of the retina and pigment epithelium of the eye in chick embryos

Undissociated tissue explants of the retina and retinal pigment epithelium (RPE) of 3,5-, 4-, 5- and 8-day-old chick embryos were cultured in vitro. After 7 days in culture, lentoids were observed in explants of either retina or RPE from 3,5-, 4- and 5-day-old embryos. As demonstrated by immunohistochemistry, these lentoids contained specific chick lens proteins (alpha-, beta- and delta-crystallins). No crystallin-containing cells were found in eye tissue explants from 8-day-old embryos. However, when 5-bromo-deoxyuridine (25 microM) was introduced into the medium at the beginning of culturing (for 12 h), large eosinophilic cells containing alpha-, beta- and delta-crystallins were detected in retinal explants of the 8-day old embryos. Thus, retina and RPE of 3,5-5-day-old chick embryos are capable of lens differentiation after explantation in vitro without dissociation into individual cells. This capacity is lost during development.     

Chick embryo retina development in vitro: The effect of insulin

In this paper we study the development of chick embryo retina culturedin vitro and the effects exerted by insulin. Retinas were removed from 7-day embryos and cultured in serum-and hormone-free medium for 7 additional days. Under these conditions retinal cells survived and underwent cholinergic differentiation, as previously ascertained by Hausman et al. (Dev. Brain Res., 1991, 59: 31–37). However, a great retardation of development was noted compared to uncultured control, 14-day retina. In fact both wet weight and DNA and protein content increased much slower than in ovo and the tubulin content decreased below even the starting value. In addition, although after 7 days in culture retinal cells were organized in identifiable layers, nevertheless the typical organization equivalent to 14-day in ovo retina was absent. The addition of insulin in the medium markedly increased the wet weight of cultured retinas, their protein content and the level of tubulin pools, particularly that of non-assembled fraction. Nevertheless insulin did not modify DNA synthesis and did not induce the increment of both neuron specific enolase and actin. Morphological observations show that insulin markedly increased the number and the thickening of the fiber layers. These results, together with the facts that retina synthesizes and secretes insulin and possesses specific insulin receptors suggest that insulin can have autocrine or paracrine regulatory functions in retinal development by exerting a general effect on retinal growth and a more specific one on tubulin production.     

The cell cycle and retinal histogenesis fidget mutant mice.

The autoradiographic method using [³H]thymidine has shown that the autosomal recessive mutant gene fidget (gene symbol fi) prolonging the presynthetic period of the cell cycle in the retinal anlage in homozygotes retards the transition of retinal cells to the differentiated state. Some retinal cells of normal embryos (+/+) start their transition to the differentiated state on the 11th day of embryogenesis, while in $\text{fi}\text{fi}$ embryos this process starts only on the 12th day. An active transition of retinal cells to the differentiated state especially in the peripheral zone of the mutant retina takes place 2 days later as compared to normal embryos. The number of differentiating cells in the retina of mutants at the stages of development studied is considerably lower as compared to the norm. The analysis of the cell cycle parameters in 15-day embryos has shown that in the mutants the retina is less mature as compared to +/+ embryos. The sequence of transition of various cell types to the differentiated state in the retina of $\text{fi}\text{fi}$ embryos is the same as in the norm. Gene fidget seems to interfere with proliferative rather than critical (quantal) cell cycles in the developing mouse retina.     

A decay of gap junctions in association with cell differentiation of neural retina in chick embryonic development.

Ultrastructural studies of thin-sectioned and freeze-cleaved materials were performed on developing retinal tissues of 3- to 9-day-old chick embryos to clarify the junctional structures between neural retinal cells and between neural retinal cells and cells of the pigmented epithelium. Frequency, size and position of gap junctions in developing neural retina are different at each stage of development. In 3-day-old embryos, some cells adhere to each other by gap junctions immediately below the outer limiting membrane of neural retinae. The size and number of gap junctions increase remarkably during 5-6 days of incubation. In this period of development, well developed gap junctions consisting of subcompartments of intramembrane particles are found between cell surfaces at both the outer limiting membrane region and the deeper portion of the neural retina. Gap junctions disappear thereafter, and at 7-5 days of incubation, small gap junctions are predominant between cell surfaces at the outer limiting membrane region, while the frequency of gap junctions in the deeper portion is very low. At 9 days of incubation, gap junctions are rarely found. Typical gap junctions are always found between neural retinal cells and those of the pigmented epithelium in embryos up to 7-5 days of incubation. Tight junctions are not found in the neural retina or between neural retina and pigmented epithelium throughout the stages examined.     

Tracing retinal fiber trajectory with a method of transposon-mediated genomic integration in chick embryo

We report convenient retinal fiber tracing by transfecting the tracer cDNA by in ovo electroporation. Long-term and stable expression of tracer proteins such as green fluorescent protein is achieved by transposon-mediated genome integration of the tracer protein expression cassette. We carried out coelectroporation of a plasmid containing CAGGS-tracer cDNA flanked by the Tol2 transposable element along with a transposase expression vector to the optic vesicle of chick embryos at stage 11. By selecting electrodes, we can label a large group of retinal ganglion cells, or a small group of retinal ganglion cells; parallel electrodes assure transfection of large areas of the retina, and needle type electrodes label small areas of the retina. The retinal fiber trajectory and terminal zone (TZ) could be detected in the precise retinotopic manner on the contra-lateral side of the optic tectum. The method has advantage in that we can show the retinal fiber trajectory in relation to the molecules that are responsible for pathfinding for the retinal fibers in the same specimen.     

银鲫转录调控因子lbh-b cDNA克隆与表达分析

Lbh (Limb-bud and heart)基因是脊椎动物中高度保守的转录调控因子, 在早期胚胎发育及某些人类疾病的发病过程中发挥着重要作用。我们前期在银鲫(Carassius gibelio)垂体转录组中筛选到一个在垂体中大量表达的基因lbh-b。为了进一步研究lbh基因在银鲫的表达特征, 首先采用RACE方法克隆了银鲫lbh基因家族的成员lbh-b基因(Cglbh-b)。Cglbh-b的cDNA全长1526 bp, 开放阅读框549 bp, 共编码182个氨基酸。生物信息学分析表明CgLbh-b蛋白与其他脊椎动物的Lbh蛋白同源性在68%以上, 可能也是无序蛋白质家族的成员之一。成体组织RT-PCR分析表明Cglbh-b仅在银鲫的垂体、端脑、卵巢及眼睛中表达。不同胚胎发育时期的表达分析表明, 在受精卵至原肠胚中Cglbh-b转录产物是以母源形式存在的mRNA, 其合子转录起始于尾芽期。胚胎整体原位杂交结果显示从受精后2d到受精后3d, Cglbh-b大量表达于脑和眼睛。此外, 随着卵子成熟Cglbh-b在银鲫垂体中的表达上调。这些结果暗示, Cglbh-b可能在调控银鲫脑和眼睛的发育以及卵子成熟过程中发挥着重要作用。     

The mouse X-linked juvenile retinoschisis cDNA: expression in photoreceptors

Retinal photoreceptor cells are particularly vulnerable to degenerations that can eventually lead to blindness. Our purpose is to identify and characterize genes expressed specifically in photoreceptors in order to increase our understanding of the biochemistry and function of these cells, and then to use these genes as candidates for the sites of mutations responsible for degenerative retinal diseases. We have characterized a cDNA, a fragment of which (SR3.1) was originally isolated by subtractive hybridization of adult, photoreceptorless rd mouse retinal cDNAs from the cDNAs of normal mouse retina. The full-length sequence of this cDNA was determined from clones obtained by screening mouse retinal and eye cDNA libraries and by using the 5'- and 3'-RACE methods. Both Northern blot analysis and in situ hybridization showed that the corresponding mRNA is expressed in rod and cone photoreceptors. The gene encoding this cDNA was mapped to the X chromosome using an interspecific cross. Based on the nucleotide and amino acid sequences, as well as chromosome mapping, we determined that this gene is the mouse ortholog (Xlrs1) of the human X-linked juvenile retinoschisis gene (XLRS1). Analysis of the predicted amino acid sequence indicates that the Xlrs1 mRNA may encode a secretable, adhesion protein. Therefore, our data suggest that X-linked juvenile retinoschisis originates from abnormalities in a photoreceptor-derived adhesion protein.     

Photopigment gene expression and rhabdom formation in the crayfish (<Emphasis Type="Italic">Procambarus clarkii</Emphasis>)

This study examines the expression of the photopigment gene in the developing retina of the freshwater crayfish Procambarus clarkii(Crustacea, Malacostraca, Decapoda). Both sense and anti-sense RNA probes were used for in situ hybridization (ISH) of whole embryos collected at various stages during development. A characteristic of retinal development is the formation of screening pigment in the retinular cells of the retinal ommatidia. This pigmentation is seen as a band that begins at the lateral side of the retinal field and progresses medially. At hatching the retina is approximately 50% pigmented. ISH of whole embryos shows that expression of the photopigment gene by the retinular cells correlates with the extent of the screening pigment band in the retina and with the presence of rhabdoms within the ommatidia. Sections taken through embryos after being hybridized indicate that staining is localized in the cytoplasm of the retinular cells and in the axonal region below the basement membrane. No staining reaction was seen in the rhabdoms of older ommatidia. ISH staining was also seen at the anterior midline of the protocerebrum where extraretinal photoreceptors have been reported. The data presented here show a close correlation of opsin expression within the retinular cells of the ommatidia and the formation of the very early rhabdoms, similar to Drosophila. The results will be discussed in relation to recent studies in Drosophila that suggest rhodopsin plays a role in effecting the organization of the terminal web-like cytoskeleton at the base of the developing rhabdom microvilli.     

Targeted disruption of Aldh1a1 (Raldh1) provides evidence for a complex mechanism of retinoic acid synthesis in the developing retina

Genetic studies have shown that retinoic acid (RA) signaling is required for mouse retina development, controlled in part by an RA-generating aldehyde dehydrogenase encoded by Aldh1a2 (Raldh2) expressed transiently in the optic vesicles. We examined the function of a related gene, Aldh1a1 (Raldh1), expressed throughout development in the dorsal retina. Raldh1(-/-) mice are viable and exhibit apparently normal retinal morphology despite a complete absence of Raldh1 protein in the dorsal neural retina. RA signaling in the optic cup, detected by using a RARE-lacZ transgene, is not significantly altered in Raldh1(-/-) embryos at embryonic day 10.5, possibly due to normal expression of Aldh1a3 (Raldh3) in dorsal retinal pigment epithelium and ventral neural retina. However, at E16.5 when Raldh3 is expressed ventrally but not dorsally, Raldh1(-/-) embryos lack RARE-lacZ expression in the dorsal retina and its retinocollicular axonal projections, whereas normal RARE-lacZ expression is detected in the ventral retina and its axonal projections. Retrograde labeling of adult Raldh1(-/-) retinal ganglion cells indicated that dorsal retinal axons project to the superior colliculus, and electroretinography revealed no defect of adult visual function, suggesting that dorsal RA signaling is unnecessary for retinal ganglion cell axonal outgrowth. We observed that RA synthesis in liver of Raldh1(-/-) mice was greatly reduced, thus showing that Raldh1 indeed participates in RA synthesis in vivo. Our findings suggest that RA signaling may be necessary only during early stages of retina development and that if RA synthesis is needed in dorsal retina, it is catalyzed by multiple enzymes, including Raldh1.     

Oriented axon outgrowth from avian embryonic retinae in culture

The neural retina of avian embryos was spread on a membrane filter and cut in any desired orientation. Strips cut across the retina of 4- to 7-day chick or 3- to 6-day quail embryos were explanted onto collagen gels. Vigorous neurite outgrowth was seen for about 3 days, by which time many neurites were 3 mm long. Horseradish peroxidase (HRP) labeling showed that the cells producing the neurites were large and formed a layer near the inner limiting membrane, indicating that the neurites in vitro were axons of retinal ganglion cells. The size of the neurite population and the regions from which neurites emerged vaired with the donor age, while most neurites sprouted from the side of the explant formerly closest to the optic fissure. This pattern closely resembled that of axon growth in the normal retina, as revealed by SEM, silver staining, and HRP labeling. Mitotic inhibitors (Ara-C and FUdR) did not alter the neurite outgrowth. Pretreatment of retinae with trypsin or collagenase did not disorganize axons at the time of explantation, but tended to equalize neurite emergence on each side of the retinal strips. We suggest that microenvironmental factors, especially the enzyme-labile inner limiting membrane, are important for axon guidance in the retina.     

ycaCR基因在文昌鱼早期胚胎表达研究

文昌鱼作为现存的与脊椎动物最接近的无脊椎动物,一直被作为研究生物进化和胚胎发育的典型材料.利用整体原位杂交方法对从文昌鱼肠cDNA文库克隆到的ycaCR基因进行基因的胚胎表达模式研究,结果显示该基因在早期胚胎发育阶段没有表达,在2天幼虫的原始消化道表达,暗示ycaCR基因可能在原始消化道内发挥生物学作用.     

Fully differentiated Xenopus eye fragments regenerate to form pattern-duplicated visuo-tectal projections

In order to determine if differentiated Xenopus retina is capable of undergoing regeneration and duplicative pattern formation, we devised a new surgical technique for removal of the temporal two-thirds of the retina. In a series of progressively older larval eyes starting with late tailbud stage embryos (stage 38) and extending to limb-bud stage tadpoles (stage 48), nasal one-third-sized eye fragments successfully regenerated to form nearly normal sized eyes over 75% of the time. Histological preparations showed that early wound healing involved the formation of a neuroepithelium at the ventro-temporal region of the fragment. The pigmented retinal epithelium and associated retinal tissue appeared to be involved in this process. Animals from each stage were reared through metamorphosis and electrophysiologic techniques were employed to determine visuo-tectal projections. Seventy percent of stage 38 animals showed evidence of pattern-duplicated projections. Ninety percent of their responding tectal points showed duplicate innervation from two retinal regions. Older animals (stages 44 to 48) showed less duplication. Only 52% of their responding tectal points duplicated (P less than 0.001). Thus, fully differentiated Xenopus retina can undergo regeneration and duplicative pattern formation similar to that shown by embryonic retinal tissue.     

The interaction of the coloboma and aphakia mutant genes in mice

The eye development has been studied in the 12-day-old, 14-day-old embryos and in neonates of Cm/+ ak/ak genotype. The gene coloboma (Cm) in heterozygous state causes a typical coloboma of the iris and the gene aphakia (ak) blocks the lens development in the homozygotes. It has been shown that in Cm/+ ak/ak mice the eyes go through mainly the same abnormal development as that in +/+ ak/ak animals. In mice of both genotypes the lens morphogenesis blocking at the vesicle stage and the retinal fold in the dorsal half of the eye develops. However, the ventral retinal fold which is characteristic for the +/+ ak/ak mice does not form in the Cm/+ ak/ak animals that is the result of the interaction of Cm and ak genes in the eye morphogenesis. The Cm gene suppressing the growth of the retina ventral half inhibits the formation of its fold in Cm/+ ak/ak embryos. As a result of the gene interaction a certain normalization of the eye development compared to the +/+ ak/ak mice is observed in the Cm/+ ak/ak animals. The obtained data show that the Cm gene expresses in the cell clones of the retina ventral half.     

Expressions of Raldh3 and Raldh4 during zebrafish early development

Retinoic acid (RA) plays crucial roles in vertebrate embryogenesis. Four retinal dehydrogenases (Raldhs) that are responsible for RA synthesis have been characterized in mammals. However, only Raldh2 ortholog is identified in zebrafish. Here, we report the identification of raldh3 and raldh4 genes in zebrafish. The predicted proteins encoded by zebrafish raldh3 and raldh4 exhibit 70.0% and 73.5% amino acid identities with mouse Raldh3 and Raldh4, respectively. RT-PCR analyses reveal that both raldh3 and raldh4 mRNAs are present in early development. However, whole mount in situ hybridization shows that raldh3 mRNA is first expressed in the developing eye region of zebrafish embryos at 10-somite stage. At 24 hpf (hours post fertilization), raldh3 mRNA is expressed in the ventral retina of eye. At 36 hpf, the mRNA is also expressed in otic vesicle in addition to ventral retina, and it maintains its expression pattern till 2 dpf (days post fertilization). At 3 dpf, raldh3 mRNA becomes very weak in ventral retina but is present in otic vesicle at a high level. At 5 dpf and 7 dpf, raldh3 is no longer expressed in eyes but is expressed in otic vesicles at a very low level. raldh4 mRNA is initially detected in developing liver and intestine regions at 2 dpf embryos. Its expression level becomes very high in these two regions of embryos from 3 dpf to 5 dpf. Analysis on the sections of 5 dpf embryos reveals that raldh4 is expressed in the epithelium of intestine. At 7 dpf, raldh4 mRNA is only weakly expressed in the epithelium of intestinal bulb.     

Proper patterning of the optic fissure requires the sequential activity of BMP7 and SHH

The optic disc develops at the interface between optic stalk and retina, and enables both the exit of visual fibres and the entrance of mesenchymal cells that will form the hyaloid artery. In spite of the importance of the optic disc for eye function, little is known about the mechanisms that control its development. Here, we show that in mouse embryos, retinal fissure precursors can be recognised by the expression of netrin 1 and the overlapping distribution of both optic stalk (Pax2, Vax1) and ventral neural retina markers (Vax2, Raldh3). We also show that in the absence of Bmp7, fissure formation is not initiated. This absence is associated with a reduced cell proliferation and apoptosis in the proximoventral quadrant of the optic cup, lack of the hyaloid artery, optic nerve aplasia, and intra-retinal misrouting of RGC axons. BMP7 addition to organotypic cultures of optic vesicles from Bmp7-/- embryos rescues Pax2 expression in the ventral region, while follistatin, a BMP7 antagonist, prevents it in early, but not in late, optic vesicle cultures from wild-type embryos. The presence of Pax2-positive cells in late optic cup is instead abolished by interfering with Shh signalling. Furthermore, SHH addition re-establishes Pax2 expression in late optic cups derived from ocular retardation (or) embryos, where optic disc development is impaired owing to the near absence of SHH-producing RGC. Collectively, these data indicate that BMP7 is required for retinal fissure formation and that its activity is needed, before SHH signalling, for the generation of PAX2-positive cells at the optic disc.     

Vax2 inactivation in mouse determines alteration of the eye dorsal-ventral axis, misrouting of the optic fibres and eye coloboma

Vax2 is a homeobox gene whose expression is confined to the ventral region of the prospective neural retina. Overexpression of this gene at early stages of development in Xenopus and in chicken embryos determines a ventralisation of the retina, thus suggesting its role in the molecular pathway that underlies eye development. We describe the generation and characterisation of a mouse with a targeted null mutation of the Vax2 gene. Vax2 homozygous mutant mice display incomplete closure of the optic fissure that leads to eye coloboma. This phenotype is not fully penetrant, suggesting that additional factors contribute to its generation. Vax2 inactivation determines dorsalisation of the expression of mid-late (Ephb2 and Efnb2) but not early (Pax2 and Tbx5) markers of dorsal-ventral polarity in the developing retina. Finally, Vax2 mutant mice exhibit abnormal projections of ventral retinal ganglion cells. In particular, we observed the almost complete absence of ipsilaterally projecting retinal ganglion cells axons in the optic chiasm and alteration of the retinocollicular projections. All these findings indicate that Vax2 is required for the proper closure of the optic fissure, for the establishment of a physiological asymmetry on the dorsal-ventral axis of the eye and for the formation of appropriate retinocollicular connections.