Cadherin-7 function in zebrafish development

Cadherin cell adhesion molecules play crucial roles in vertebrate development. Most studies have focused on examining the functions of classical type I cadherins (e.g., cadherin-2) in the development of vertebrates. Little information is available concerning the function of classical type II cadherins (e.g., cadherin-7) in vertebrate development. We have previously shown that cadherin-7 mRNA exhibits a dynamic expression pattern in the central nervous system and notochord in embryonic zebrafish. To gain insight into the role of cadherin-7 in the formation of these structures, we analyzed their formation in zebrafish embryos injected with cadherin-7-specific antisense morpholino oligonucleotides (MO). Notochord development was severely disrupted in MO-injected embryos, whereas gross defects in the development of the central nervous system were not detected in MO-injected embryos. Our results thus demonstrate that cadherin-7 plays an important role in the normal development of the zebrafish notochord.     

参与小鼠神经发育的mCcd1基因的组织表达

介绍了一种可能在神经发育过程中起重要调节作用的基因mCcd1在小鼠发育过程中的mRNA和蛋白质水平的表达变化。通过对新生和成年小鼠多组织免疫杂交(Western Blot)和反转录PCR(RT-PCR)检测发现:该基因在多种组织中广泛表达,但脑部显示高表达,并且新生鼠各组织表达均高于成体。小鼠11.5 d胚胎切片免疫组化实验也支持这一结果。mCcd1在神经系统发育早期的表达暗示它可能参与了神经系统的发育过程。     

Identification of a Drosophila homologue of alpha-catenin and its association with the armadillo protein

The cadherin cell adhesion system plays a central role in cell-cell adhesion in vertebrates, but its homologues are not identified in the invertebrate. alpha-Catenins are a group of proteins associated with cadherins, and this association is crucial for the cadherins' function. Here, we report the cloning of a Drosophila alpha-catenin gene by low stringent hybridization with a mouse alpha E-catenin probe. Isolated cDNAs encoded a 110-kD protein with 60% identity to mouse alpha E- catenin, and this protein was termed D alpha-catenin. The gene of this protein was located at the chromosome band 80B. Immunostaining analysis using a mAb to D alpha-catenin revealed that it was localized to cell- cell contact sites, expressed throughout development and present in a wide variety of tissues. When this protein was immunoprecipitated from detergent extracts of Drosophila embryos or cell lines, several proteins co-precipitated. These included the armadillo product which was known to be a Drosophila homologue of beta-catenin, another cadherin-associated protein in vertebrates, and a 150-kD glycoprotein. These results strongly suggest that Drosophila has a cell adhesion machinery homologous to the vertebrate cadherin-catenin system.     

Expression of murine early embryonic antigens, SSEA-1 and antigenic determinant of EMA-1, in embryos and ovarian follicles of a teleost medaka (Oryzias latipes)

Stage-specific embryonic antigen-1 (SSEA-1) and the antigenic determinant of monoclonal antibody EMA-1 are expressed in a stage-specific manner in mouse early embryos. To study whether these antigens generally exist in fish, expression of the antigens was examined in embryos, ovarian follicles, and adult tissues of a teleost medaka (Oryzias latipes), using immunohistochemical techniques. In 1-cell-stage embryos, these carbohydrate antigens were found in numerous cytoplasmic granules in the blastodisc and the cortical cytoplasm. These granules gradually decreased in number as the embryos developed. In 4-cell-stage embryos, the antigens appeared on the cleavage planes and were located on the cleavage planes within the blastoderm in the following cleavage stages. In blastula-stage embryos, the expression was ubiquitously found on the cell surface of blastomeres. At the mid-gastrula stage, the antigens were restricted to the enveloping layer, yolk syncytial layer, and cortical cytoplasm, but were rarely found in deep cells that contribute to formation of the embryonic body. In later-stage embryos and adult fish, the antigens were located in various tissues. In ovarian follicles, the antigens were found in granules of oocytes and granulosa cells. These observations were basically consistent with those in mice; however, expression in 1-cell-stage embryos and ovarian follicles has not been observed in mice. This unexpected finding suggests that the antigens are produced in granulosa cells and transferred to 1-cell-stage embryos via oocytes, and that the antigens involved in the early developmental process are maternally prepared in teleosts.     

Monoclonal antibodies to laminin reveal the heterogeneity of basement membranes in the developing and adult mouse tissues

Two monoclonal antibodies raised against laminin isolated from a mouse parietal yolk sac cell line were used for immunohistochemical studies of basement membranes of the mouse embryo and various fetal and adult tissues. No immunoreactivity with either of the two monoclonal antibodies could be detected in the preimplantation-stage embryos, although it has been shown that these embryos contain extracellular laminin reactive with the conventional polyclonal antilaminin antibodies. Reichert's membrane in early postimplantation stages of development reacted with the monoclonal antibody LAM-I but not with the antibody LAM-II. However, from day 8 of pregnancy onward the Reichert's membrane reacted with both antibodies. Basement membranes of the embryo proper were unreactive with both monoclonal antibodies until day 12 of pregnancy. By day 14 some basement membranes of the fetal tissues became reactive with one or both monoclonal antibodies, whereas others remained still unreactive. In the 17-d fetus and the newborn mouse most of the basement membranes reacted with both monoclonal antibodies, whereas others still reacted with only one. Similar heterogeneity in the immunoreactivity of basement membranes of various tissues was noted in the adult mouse as well. These results indicate that the immunoreactivity of laminin in the extracellular matrix changes during development and that the basement membranes in various anatomic locations display heterogeneity even in the adult mouse.     

Cadherins in neuronal morphogenesis and function

Classic cadherins represent a family of calcium-dependent homophilic cell–cell adhesion molecules. They confer strong adhesiveness to animal cells when they are anchored to the actin cytoskeleton via their cytoplasmic binding partners, catenins. The cadherin/catenin adhesion system plays key roles in the morphogenesis and function of the vertebrate and invertebrate nervous systems. In early vertebrate development, cadherins are involved in multiple events of brain morphogenesis including the formation and maintenance of the neuroepithelium, neurite extension and migration of neuronal cells. In the invertebrate nervous system, classic cadherin-mediated cell–cell interaction plays important roles in wiring among neurons. For synaptogenesis, the cadherin/catenin system not only stabilizes cell–cell contacts at excitatory synapses but also assembles synaptic molecules at synaptic sites. Furthermore, this system is involved in synaptic plasticity. Recent studies on the role of individual cadherin subtypes at synapses indicate that individual cadherin subtypes play their own unique role to regulate synaptic activities.     

Expression of Catenins during Mouse Embryonic Development and in Adult Tissues

Classical cadherins are cell-surface glycoproteins that mediate calcium-dependent cell adhesion. The cytoplasmic domain of these glycoproteins is linked to the cytoskeleton through the catenins (α, β and γ). The catenins are intracellular polypeptides that are part of a complex sub-membranous network modulating the adhesive ability of the cells. One approach to elucidate the role of these molecules in the cell is to investigate their distribution during mouse development and in adult tissues. This study reports that catenins are widely expressed but in varying amounts in embryos and adult tissues. The expression of all three catenins is most prominent in the adult heart muscle and in epithelia of all developmental stages. In other embryonic and adult tissues, lower expression of catenins was detected, e.g., in smooth muscle or connective tissue. Catenins are coexpressed with various cadherins in different tissues. Gastrulation is the first time during embryogenesis when a discrepancy occurs between the expression of catenins and E-cadherin. E-cadherin expression is suppressed in mesodermal cells but not the expression of catenins. This discrepancy suggests that another cadherin may interact with catenins. Similarly, E-cadherin is generally expressed in adult liver but not in the regions surrounding the central veins. In contrast, catenins are uniformly expressed in the liver, suggesting that they are associated with other cadherins in E-cadherin negative cells. Finally, the three catenins are not always concurrently expressed. For example, in peripheral nerves, only β-catenin is observable, and in smooth muscle plakoglobin is not detectable.     

Expression of ORAI1, a Plasma Membrane Resident Subunit of the CRAC Channel,in Rodent and Non-rodent Species

We determined the expression of ORAI1 protein in rodent and non-rodent tissues using a monoclonal antibody directed against an extracellular loop of the protein. Previous reports using antibodies directed at the C-terminus of ORAI1 have not detected central nervous system (CNS) expression. Our results demonstrate broad tissue expression that includes the CNS using a unique monoclonal antibody specific to an extracellular loop of ORAI1. In addition, we present in situ hybridization (ISH) results using a probe within the middle of the mouse coding region showing CNS expression of Orai1 RNA. We contrast the patterns of rodent and human tissue expression and conclude that rodents have similar expression of ORAI1 in most tissue types when compared to primates, with an important exception being the male reproductive system, where human-specific expression is observed.     

Drosophila non-muscle alpha-actinin is localized in nurse cell actin bundles and ring canals, but is not required for fertility

The single copy Drosophila alpha-actinin gene is alternatively spliced to generate three different isoforms that are expressed in larval muscle, adult muscle and non-muscle cells, respectively. We have generated novel alpha-actinin alleles, which specifically remove the non-muscle isoform. Homozygous mutant flies are viable and fertile with no obvious defects. Using a monoclonal antibody that recognizes all three splice variants, we compared alpha-actinin distribution in wild type and mutant embryos and ovaries. We found that non-muscle alpha-actinin was present in young embryos and in the embryonic central nervous system. In ovaries, non-muscle alpha-actinin was localized in the nurse cell subcortical cytoskeleton, cytoplasmic actin cables and ring canals. In the mutant, alpha-actinin expression remained in muscle tissues, but also in a subpopulation of epithelial cells in both embryos and ovaries. This suggests that various populations of non-muscle cells regulate alpha-actinin expression in different ways. We also show that ectopically expressed adult muscle-specific alpha-actinin localizes to all F-actin containing structures in the nurse cells in the absence of endogenous non-muscle alpha-actinin.     

Spatial aspects of neural induction in Xenopus laevis

A monoclonal antibody, 2G9, has been identified and characterised as a marker of neural differentiation in Xenopus. The epitope is present throughout the adult central nervous system and in peripheral nerves. Staining is first detected in embryos at stage 21 in the thoracic region. By stage 29 it stains the whole central nervous system, except the tail tip. The epitope is present in a 65K Mr protein, and includes sialic acid. The antibody also reacts with neural tissue in mice and axolotls and newts. 2G9 was used to show that both notochord and somites are capable of neural induction, and the stimulus is present as late as stage 22. Attempts to demonstrate the induction of nervous system by developing nervous system (homoiogenetic induction) were unsuccessful. The view that the lateral extent of the nervous system might be determined by that of the inductive stimulus is discussed. Neural induction was detected as early as stage 10 and occurs in embryos without gastrulation and without cell division from stage 7 1/2.     

Reactivity of a Monoclonal Antibody Raised against Human Leukemic Cells to Embryonic and Adult Tissues of the Mouse and Teratocarcinomas

C-9-1, a monoclonal IgM antibody raised against human null cell acute lymphocytic leukemia cells reacted with restricted regions of embryonic and adult tissues of the mouse. The antigen positive sites in the embryos included embryonic ectoderm, visceral endoderm, trophoblastic cells invading the maternal decidua of 5∼7-day embryos, primordial germ cells of 10∼12-day embryos, epithelium of nasal chamber, the bronchus, Mullerian duct, epididymis and bladder of 12∼17-day embryos. In the adult mice, C-9-1 antigen was detected in renal tubules, a part of stomach, bladder, endometrium and epididymal sperm. Embryonal carcinoma cells, but not endodermal cells of teratocarcinoma expressed the antigen. Thus, C-9-1 antigen showed distribution similar to SSEA-1. However, C-9-1 antigen was not detected in preimplantation embryos, nor in oviduct, both of which are positive for SSEA-1.     

Dmbx1, a novel evolutionarily conserved paired-like homeobox gene expressed in the brain of mouse embryos.

To identify novel homeobox genes expressed during mouse embryogenesis, we searched the databases and found a novel mouse paired-like homeobox gene, Dmbx1(diencephalon/mesencephalon-expressed brain homeobox gene 1), that is also conserved in zebrafish and human. Linkage analysis mapped mouse Dmbx1 to the mid-portion of chromosome 4 that is the homologous gene cluster region of human chromosome 1, where human DMBX1 is located. Both mouse and human Dmbx1/DMBX1 have four coding exons and their gene structures are conserved. Whole-mount in situ hybridization revealed that Dmbx1 expression is detected in 7.5-9.5 dpc mouse embryos. At 7.5 and 8.5 dpc, Dmbx1 is expressed in a sub-region of the anterior head folds. At 9.5 dpc, expression is observed in the caudal diencephalon as well as in the mesencephalon and is restricted to the neuroepithelium. Expression in adult tissues was detected in brain, stomach, and testis. Dmbx1 provides a unique marker of the developing anterior nervous system and should provide a useful molecular resource to elucidate the mechanisms that pattern the vertebrate brain.     

A novel cadherin cell adhesion molecule: its expression patterns associated with implantation and organogenesis of mouse embryos

The Ca2+-dependent cell adhesion molecules, termed cadherins, were previously divided into two subclasses, E- and N-types, with different adhesive specificity. In this study, we identified a novel class of cadherin, termed P-cadherin, using a visceral endoderm cell line PSA5- E. This cadherin was a 118,000-D glycoprotein and distinct from E- and N-cadherins in immunological specificity and molecular mass. In accord with these findings, cells with P-cadherin did not cross-adhere with cells with E-cadherin. P-Cadherin first appeared in developing mouse embryos in the extraembryonic ectoderm and the visceral endoderm at the egg cylinder stage and later was expressed in various tissues. The placenta and the uterine decidua most abundantly expressed this cadherin. The expression of P-cadherin was transient in many tissues, and its permanent expression was limited to certain tissues such as the epidermis, the mesothelium, and the corneal endothelium. When the tissue distribution of P-cadherin was compared with that of E-cadherin, we found that: each cadherin displayed a unique spatio-temporal pattern of expression; P-cadherin was co-expressed with E-cadherin in local regions of various tissues; and onset or termination of expression of P- cadherin was closely associated with connection or segregation of cell layers, as found with other cadherins. These results suggested that differential expression of multiple classes of cadherins play a role in implantation and morphogenesis of embryos by providing cells with heterogenous adhesive specificity.     

Cadherin-1, -2 and -4 expression in the cranial ganglia and lateral line system of developing zebrafish

Cadherins are cell adhesion molecules that play important roles in development of a variety of tissues and organs including the nervous system. In this study we analyzed expression patterns of three zebrafish classical (type I) cadherins (cadherin-1, -2, and -4) in the embryonic zebrafish cranial ganglia and lateral line system using in situ hybridization and immunohistochemical methods. All three cadherins exhibit distinct spatiotemporal patterns of expression during cranial ganglia and lateral line system development. cadherin-1 message was detected in the trigeminal and facial ganglia, in the lateral line ganglia, and in most of neuromasts in the lateral lines. Cadherin-2 mRNA and protein were expressed by the majority of the cranial ganglia and lateral line system. Both cadherins were found in embryos younger than 24 hours post fertilization as well as in 2-3-day old embryos and larvae. In contrast, cadherin-4 mRNA and protein expression was detected in embryos older than 30 hours post fertilization and limited to the trigeminal, statoacoustic, and vagal cranial ganglia, and the lateral line ganglia of older embryos and larvae.     

The oocyte lamin persists as a single major component of the nuclear lamina during embryonic development of the surf clam

Nuclei and nuclear lamina-enriched fractions, isolated from 1 to 5-day-old embryos of the surf clam, Spisula solidissima, contain only one major lamin protein, which appears to be identical to the oocyte lamin (L67), as judged by 2D IEF/SDS PAGE, reactivity with a polyclonal antibody directed against L67 and 125I tryptic peptide mapping. The same protein is also present in liver, muscle, nerve and testis from adult animals. No proteins--recognized by several poly- and monoclonal antibodies, specific for somatic lamins from different vertebrate species or the oocyte lamin LIII of Xenopus- have been detected in nuclei or NL-enriched preparations, isolated from embryos or adult tissues. Synthesis of L67 is detectable in embryos 2h after fertilization; it reaches a maximum in 6h-old embryos and gradually declines thereafter. These results argue that the composition of the NL bears no obvious relationship to the structural and functional changes that take place during the embryonic development of this invertebrate.