Homeobox Genes Expressed During Echinoderm Arm Regeneration

Regeneration in echinoderms has proved to be more amenable to study in the laboratory than the more classical vertebrate models, since the smaller genome size and the absence of multiple orthologs for different genes in echinoderms simplify the analysis of gene function during regeneration. In order to understand the role of homeobox-containing genes during arm regeneration in echinoderms, we isolated the complement of genes belonging to the Hox class that are expressed during this process in two major echinoderm groups: asteroids (Echinaster sepositus and Asterias rubens) and ophiuroids (Amphiura filiformis), both of which show an extraordinary capacity for regeneration. By exploiting the sequence conservation of the homeobox, putative orthologs of several Hox genes belonging to the anterior, medial, and posterior groups were isolated. We also report the isolation of a few Hox-like genes expressed in the same systems.     

Regeneration of the Cornea in Adult Newts: Overall Process and Behavior of Epithelial Cells

Regeneration of the cornea in adult newts was studied by means of light- and electron-microscopic techniques. We focused our analysis particularly on the behavior of epithelial cells during the initial process of wound healing after we had excised a central disk about 0.5 mm in diameter through the entire thickness of the cornea. Fine fibrous material, assumed to be fibrin, appeared within 30 min to form an acellular layer of mucous consistency which sealed the wound opening completely. The cut edge of corneal epithelium moved centripetally on this layer by coordinate movement of individual epithelial cells. Almost all cells of the remained epithelium were completely rearranged within 5 h after excision. Some desmosomes among the epithelial cells persisted during the process of cellular rearrangement. Thus, the wound opening was covered completely within 24 h by the epithelium alone without cell proliferation. Cytochalasin B or D completely inhibited movement of the corneal epithelium on the stroma in conditions in vitro, suggesting active participation of intracellular contractile microfilaments in such movement of the epithelium. Active growth of cells in the epithelium started on day 3 and the epithelium recovered its normal thickness by day 10 after excision.
After the recovery of the epithelium, keratocytes moved out from the wounded edge of the remained corneal stroma. These keratocytes actively proliferated in the wound area under the newly formed epithelium and participated in the stromal reconstitution, which proceeded gradually for more than 5 weeks.     

Noggin Expression in the Adult Retina Suggests a Conserved Role during Vertebrate Evolution

Vertebrates share common mechanisms in the control of development and in the maintenance of neural and retinal function. The secreted factor Noggin, a BMP inhibitor, plays a crucial role in neural induction during embryonic development. Moreover, we have shown its involvement in retinal differentiation of pluripotent cells. Here we show Noggin expression in the adult retina in three vertebrate species. Four Noggin genes are present in zebrafish (Danio rerio; ZbNog1, 2, 3, 5), three in frog (Xenopus laevis; XenNog1, 2 and 4), and one in mouse (Mus musculus; mNog). Quantitative RT-PCR experiments show the presence of ZbNog3 and ZbNog5 mRNAs, but not ZbNog1 and ZbNog2, in the adult zebrafish retina. All three genes are expressed in the frog retina, and mNog in the mouse. Immunohistochemistry data show that Noggin proteins are predominantly localized in the Golgi apparatus of photoreceptors and in the fibers of the outer plexiform layer. Lower expression levels are also found in inner plexiform layer fibers, in ganglion cells, in the ciliary marginal zone, and in retinal pigmented epithelium. Our results show that Noggin has a specific cellular and sub-cellular expression in the adult vertebrate retina, which is conserved during evolution. In addition to its established role during embryonic development, we postulate that Noggin also exerts a functional role in the adult retina.     

Evolution of Antennapedia-Class Homeobox Genes

Antennapedia (Antp)-class homeobox genes are involved in the determination of pattern formation along the anterior-posterior axis of the animal embryo. A phylogenetic analysis of Antp-class homeodomains of the nematode, Drosophila, amphioxus, mouse, and human indicates that the 13 cognate group genes of this gene family can be divided into two major groups, i.e., groups I and II. Group I genes can further be divided into subgroups A (cognate groups 1-2), B (cognate group 3), and C (cognate groups 4-8), and group II genes can be divided into subgroups D (cognate groups 9-10) and E (cognate groups 11-13), though this classification is somewhat ambiguous. Evolutionary distances among different amino acid sequences suggest that the divergence between group I and group II genes occurred ~1000 million years (MY) ago, and the five different subgroups were formed by ~600 MY ago, probably before the divergence of Pseudocoelomates (e.g., nematodes) and Coelomates (e.g., insects and chordates). Our results show that the genes that are phylogenetically close are also closely located in the chromosome, suggesting that the colinearity between the gene expression and gene arrangement was generated by successive tandem gene duplications and that the gene arrangement has been maintained by some sort of selection.     

Melatonin Signaling Modulates Clock Genes Expression in the Mouse Retina

Previous studies have shown that retinal melatonin plays an important role in the regulation of retinal daily and circadian rhythms. Melatonin exerts its influence by binding to G-protein coupled receptors named melatonin receptor type 1 and type 2 and both receptors are present in the mouse retina. Earlier studies have shown that clock genes are rhythmically expressed in the mouse retina and melatonin signaling may be implicated in the modulation of clock gene expression in this tissue. In this study we determined the daily and circadian expression patterns of Per1, Per2, Bmal1, Dbp, Nampt and c-fos in the retina and in the photoreceptor layer (using laser capture microdissection) in C3H-f+/+ and in melatonin receptors of knockout (MT₁ and MT₂) of the same genetic background using real-time quantitative RT-PCR. Our data indicated that clock and clock-controlled genes are rhythmically expressed in the retina and in the photoreceptor layer. Removal of melatonin signaling significantly affected the pattern of expression in the retina whereas in the photoreceptor layer only the Bmal1 circadian pattern of expression was affected by melatonin signaling removal. In conclusion, our data further support the notion that melatonin signaling may be important for the regulation of clock gene expression in the inner or ganglion cells layer, but not in photoreceptors.     

Analysis of the Expression Pattern of Regulatory Genes Pax6, Prox1, and Six3 during Regeneration of Eye Structures in the Newt

We studied tissue-specific expression of homeobox genes Pax6, Prox1, and Six3 during regeneration of the retina and lens. In the native retina, mRNA of Pax6, Prox1, and Six3 was predominantly localized in ganglion cells and in the inner nuclear layer of the retina. Active Pax6, Prox1, and Six3 expression was detected at early stages of regeneration in all proliferating neuroblasts forming the retinal primordium. Low levels of Pax6, Prox1, and Six3 mRNA were revealed in depigmented cells of the pigment epithelium as compared to the proliferating neuroblasts. At the intermediate stage of retinal regeneration, the distribution of Pax6, Prox1, and Six3 mRNA was diffuse and even all over the primordium. During differentiation of the cellular layers in the course of retinal regeneration, Pax6, Prox1, and Six3 mRNA was predominantly localized in ganglion cells and in the inner part of the inner nuclear layer, which was similar to the native retina. An increased expression was revealed in the peripheral regenerated retina where multipotent cells were localized. The dual role of regulatory genes Pax6, Prox1, and Six3 during regeneration of eye structures has been revealed; these genes controlled cell proliferation and subsequent differentiation of ganglion, amacrine, and horizontal cells. High hybridization signal of all studied genes was revealed in actively proliferating epithelial cells of the native and regenerating lens, while the corneal epithelium demonstrated a lower signal. Pax6 and Prox1 expression was also revealed in single choroid cells of the regenerating eye.     

Homeobox Genes in the Developing Mouse Brain

Abstract: Any list of past and recent findings on vertebrate brain prenatal development would have to include the fundamental roles of homeobox genes, the genes encoding the nuclear regulatory homeodomain proteins. The discovery of homeobox genes and their involvement as master regulatory elements in programing the development of an embryo into a complete adult organism has provided a key to our understanding of ontogenesis. Also, the correlation of mouse developmental mutants and their corresponding human syndromes with mutations in homeobox genes has provided further evidence for the fundamental role of homeobox genes during the vertebrate brain embryonic development. Here, we review the expression patterns and the phenotypes of gene mutations that implicate a large repertoire of mouse homeobox genes in the specification of neuronal functions during brain embryogenesis.     

植物同源异型枢基因的研究进展

植物同源异型枢基因是涉及到植物个体发育的一类重要转录因子编码基因.这类基因及其编码蛋白的结构具有明显的保守性,在植物中广泛存在,研究这类基因,对于揭示植物的发育机制具有重要意义.     

N-Acetyl-Glucosaminidase Activity during Limb Regeneration in the Adult Newt

N-Acetyl-glucosaminidase activity was measured during the first 25 days of limb regeneration. It was found that the enzyme is present in the normal limb. Following amputation a significant drop was obtained at day 3. A significant increase in enzyme activity was found at day 5 followed by a second drop by day 10. For days 12–15 a second peak of enzyme activity was detected, followed by a third drop; by day 25, normal levels of enzyme activity were detected. Histochemical localization of the enzyme in tissue samples showing enzyme activity as detected biochemically (days 5 and 17 of regeneration) gave negative results. However, enzyme activity was found in the incubation medium, indicating that the enzyme is released from the cells. The peaks of enzyme activity coincide with the stages of limb regeneration where a high degree of tissue demolition and cell lysis occurs. The latter are important events in the regeneration process, cell dedifferentiation, and blastema formation.     

Expression of the Homeobox Genes OTX2 and OTX1 in the Early Developing Human Brain

In rodents, the Otx2 gene is expressed in the diencephalon, mesencephalon, and cerebellum and is crucial for the development of these brain regions. Together with Otx1, Otx2 is known to cooperate with other genes to develop the caudal forebrain and, further, Otx1 is also involved in differentiation of young neurons of the deeper cortical layers. We have studied the spatial and temporal expression of the two homeobox genes OTX2 and OTX1 in human fetal brains from 7 to 14 weeks postconception by in situ hybridization and immunohistochemistry. OTX2 was expressed in the diencephalon, mesencephalon, and choroid plexus, with a minor expression in the basal telencephalon. The expression of OTX2 in the hippocampal anlage was strong, with no expression in the adjacent neocortex. Contrarily, the OTX1 expression was predominantly located in the proliferative zones of the neocortex. At later stages, the OTX2 protein was found in the subcommissural organ, pineal gland, and cerebellum. The early expression of OTX2 and OTX1 in proliferative cell layers of the human fetal brain supports the concept that these homeobox genes are important in neuronal cell development and differentiation: OTX1 primarily in the neocortex, and OTX2 in the archicortex, diencephalon, rostral brain stem, and cerebellum. (J Histochem Cytochem 58:669–678, 2010)     

Changes in the Distribution of Fibronectin During Limb Regeneration in Newts Using Immunocytochemistry

The distribution of fibronectin in regenerating newt limbs was studied using immunocytochemistry. At appropriate intervals after the initial amputation at the elbow (10–30 days), animals were reamputated at the shoulder and processed for light microscopy. The peroxidase-antiperoxidase technique was used to localize affinity-purified antibodies to fibronectin in limb tissues. At the amputation site, fibronectin was associated with basal laminae and connective tissues adjacent to dedifferentiating limb tissues destined to form the regeneration blastema. Accumulation and growth of the blastema was accompanied by the apparent de novo synthesis of fibronectin, where it appeared randomly in the interstitium between blastemal cells. The onset of chondrogenesis was characterized by a central condensation of prechondroblasts that formed the cartilage anlagen. Fibronectin formed an amorphous network between presumptive chondroblasts. As the mature cartilage phenotype was expressed and chondrocytes became isolated in lacunae, fibronectin was greatly reduced and then disappeared. The extracellular matrix surrounding undifferentiated blastemal cells still contained fibronectin. Fibronectin was also found in high concentrations between differentiating myoblasts. A condensation of fibronectin was also observed beneath the epidermis at the distal limb tip at the onset of digit formation. These observations are consistent with the hypothesis that fibronectin may play a key role in the morphogenetic events that result in the spatial organization and subsequent differentiation of cells during pattern formation in the regenerating limb.     

Midkine-a Protein Localization in the Developing and Adult Retina of the Zebrafish and Its Function During Photoreceptor Regeneration

Midkine is a heparin binding growth factor with important functions in neuronal development and survival, but little is known about its function in the retina. Previous studies show that in the developing zebrafish, Midkine-a (Mdka) regulates cell cycle kinetics in retinal progenitors, and following injury to the adult zebrafish retina, mdka is strongly upregulated in Müller glia and the injury-induced photoreceptor progenitors. Here we provide the first data describing Mdka protein localization during different stages of retinal development and during the regeneration of photoreceptors in adults. We also experimentally test the role of Mdka during photoreceptor regeneration. The immuno-localization of Mdka reflects the complex spatiotemporal pattern of gene expression and also reveals the apparent secretion and extracellular trafficking of this protein. During embryonic retinal development the Mdka antibodies label all mitotically active cells, but at the onset of neuronal differentiation, immunostaining is also localized to the nascent inner plexiform layer. Starting at five days post fertilization through the juvenile stage, Mdka immunostaining labels the cytoplasm of horizontal cells and the overlying somata of rod photoreceptors. Double immunolabeling shows that in adult horizontal cells, Mdka co-localizes with markers of the Golgi complex. Together, these data are interpreted to show that Mdka is synthesized in horizontal cells and secreted into the outer nuclear layer. In adults, Mdka is also present in the end feet of Müller glia. Similar to mdka gene expression, Mdka in horizontal cells is regulated by circadian rhythms. After the light-induced death of photoreceptors, Mdka immuonolabeling is localized to Müller glia, the intrinsic stem cells of the zebrafish retina, and proliferating photoreceptor progenitors. Knockdown of Mdka during photoreceptor regeneration results in less proliferation and diminished regeneration of rod photoreceptors. These data suggest that during photoreceptor regeneration Mdka regulates aspects of injury-induced cell proliferation.     

Characterization of Light Lesion Paradigms and Optical Coherence Tomography as Tools to Study Adult Retina Regeneration in Zebrafish

Light-induced lesions are a powerful tool to study the amazing ability of photoreceptors to regenerate in the adult zebrafish retina. However, the specificity of the lesion towards photoreceptors or regional differences within the retina are still incompletely understood. We therefore characterized the process of degeneration and regeneration in an established paradigm, using intense white light from a fluorescence lamp on swimming fish (diffuse light lesion). We also designed a new light lesion paradigm where light is focused through a microscope onto the retina of an immobilized fish (focused light lesion). Focused light lesion has the advantage of creating a locally restricted area of damage, with the additional benefit of an untreated control eye in the same animal. In both paradigms, cell death is observed as an immediate early response, and proliferation is initiated around 2 days post lesion (dpl), peaking at 3 dpl. We furthermore find that two photoreceptor subtypes (UV and blue sensitive cones) are more susceptible towards intense white light than red/green double cones and rods. We also observed specific differences within light lesioned areas with respect to the process of photoreceptor degeneration: UV cone debris is removed later than any other type of photoreceptor in light lesions. Unspecific damage to retinal neurons occurs at the center of a focused light lesion territory, but not in the diffuse light lesion areas. We simulated the fish eye optical properties using software simulation, and show that the optical properties may explain the light lesion patterns that we observe. Furthermore, as a new tool to study retinal degeneration and regeneration in individual fish in vivo, we use spectral domain optical coherence tomography. Collectively, the light lesion and imaging assays described here represent powerful tools for studying degeneration and regeneration processes in the adult zebrafish retina.     

Abnormal Limb Regeneration without Tumor Production in Adult Newts Directed by Carcinogens, 20-Methylcholanthrene and Benzo (α) pyrene

The effects of potent carcinogens, 20-methylcholanthrene (MC) and benzo(α)pyrene (BP), on limb regeneration were studied in adult newts. A microcrystal of these carcinogens was administered directly to the blastema of forelmibs on day 7 after amputation. The formation of the regeneration cone was delayed and the cone was shifted in abnormal polarity depending upon the site of micro-crystal administration. These carcinogens affected morphogenesis of skeletal structures of regenerating limbs. Subregeneration and superregeneration of either or both carpals and digits, absence of either or both ulna and radius, and accessory limb formation have been recorded as abnormalities caused by these carcinogens. Non-carcinogenic benzocompounds did not show such effects as those of MC and BP. The regeneration blastema of the limb appears to be resistant to carcinogenic effects of the carcinogens used since tumor formation has never been observed in our study so far.     

细胞连接相关基因在大鼠肝再生中表达模式

细胞连接是组织、器官形成的基础。为在基因转录水平了解紧密连接、粘附连接、粘着斑和间隙连接相关基因在肝再生中作用,本文用搜集网站资料和查阅相关论文等方法获得上述基因,用Rat Genome 230 2.0芯片检测它们在大鼠再生肝中表达情况,将3次检验结果相同或相似、在肝再生中发生有意义表达变化、真手术组和假手术组表达差异显著的基因视为肝再生相关基因。初步证实上述4种细胞连接中79、53、109和53个基因与肝再生相关。其中,肝再生启动(部分肝切除后0.5～4h)、G0/G1过渡(PH后4～6h)、细胞增殖(部分肝切除后6～66h)、细胞分化和组织结构功能重建(部分肝切除后72～168h)等4个阶段起始表达的基因数和基因的总表达次数为124、43、122、10和249、145、957、306。表明相关基因主要在肝再生启动阶段起始表达,在不同阶段发挥作用。它们共上调972次,下调540次,表明肝再生中大多数细胞连接相关基因表达加强,少数基因表达降低。它们表达的相似性分为均上调、上调占优势、均下调、下调占优势、上调和下调相近等5类,涉及102、38、73、27和16个基因,它们表达的时间相关性分为0.5和1h、2h、4和6h、8和12h、16h、18和48h、24h、30和42h、36h、54和60h、66和72h、96h、120h、144和168h等14组,表明肝再生中细胞生理生化活动具有阶段性。它们的表达模式分为41类,表明肝再生中细胞生理生化活动具有多样性和复杂性。根据肝再生中基因表达变化和表达模式推测,肝再生早期和前期间隙连接形成增强,晚中期和后期间隙连接形成减少;早期、前期和后期粘着斑形成增强;紧密连接和粘附连接的形成贯穿于整个肝再生。