The potassium channel MBK1 (Kv1.1) is expressed in the mouse retina

1. The neurons of the retina have electrical properties that are different from those of most of the other neurons of the central nervous system. To identify the voltage-gated ion channels found in the retina, we screened mouse retinal cDNA libraries with oligonucleotide probes homologous to the mammalian K+ channel MBK1 (Kv1.1) and ligated two partial clones to produce a full-length clone with no significant differences from MBK1. 2. Expression of MBK1 mRNA was determined by RNAse protection. MBK1 mRNA was detected in retinal RNA and was also detected in brain, liver, and heart RNAs. 3. We transcribed the full-length clone, injected it into oocytes of Xenopus laevis, and measured the membrane currents 2 to 6 days later. Depolarization from a holding voltage of -90mV induced a slowly activated outward current with a peak value as large as 20 microA. The current inactivated very slowly with a single exponential time course [mean time constant, 6.5 +/- 0.4 sec (SEM) for activation voltage of -10mV]. 4. The outward current was reduced to half-maximal by 0.42 mM tetraethylammonium, 1.1 mM 4-aminopyridine, and 3.2 mM Ba2+ but was not significantly attenuated by Co2+ (1 mM). 5. The reversal potential (measured with tail currents) changed by 53mV per decade change of [K+] from 1 to 77 mM. 6. The voltage for half-maximal activation of the conductance was -26.6mV (+/- 1.7mV), and the voltage required for an e-fold increase in conductance was 6.9mV (+/- 0.5mV). 7. Thus, the mRNA for MBK1 found in the mouse retina causes the expression of a voltage-dependent K+ current which has properties suitable for may retinal neurons.     

c-Flip(L) is expressed in undifferentiated mouse male germ cells

Apoptosis represents a fundamental process during fetal/post-natal testis development. Therefore pro- and anti-apoptotic proteins are essential to regulate testis physiology. c-Flip(L) is a known inhibitor of caspase 8/10 activity; in this study its perinatal expression in mouse male germ cells was investigated. In testis sections and seminiferous tubule whole mount c-Flip(L) was found to be expressed in undifferentiated spermatogonia and to co-localize with germ stem cells markers. In vivo investigations in the vitamin-A deficient mouse, lacking differentiated germ cells, confirmed c-Flip(L) expression in undifferentiated spermatogonia. Further analyses showed Fas expression but no significant caspase 8/10 activity when c-Flip(L) was highly expressed. Altogether these data suggest that c-Flip may control the survival rate of undifferentiated spermatogonia.     

Murine embryonic antigen (SSEA-1) is expressed on human cells and structurally related human blood group antigen I is expressed on mouse embryos

The stage-specific embryonic antigen (SSEA-1), present on embryonal carcinoma cells and on murine preimplantation embryos, is defined by a monoclonal antibody. The antigenic determinant of SSEA-1 is a carbohydrate structurally related to the human blood group antigen I. Since it has been suggested that the I antigen might represent a precursor or SSEA-1, we used antibodies to SSEA-1 and to I to analyze their expression on mouse preimplantation embryos. Both are expressed on mouse embryos; moreover, I is expressed on earlier embryos than SSEA-1. The I antigen is defined by its expression on human erythrocytes; accordingly, we examined expression of I and SSEA-1 on human peripheral blood elements. We find SSEA-1 to be expressed exclusively on human granulocytes while I is found only on erythrocytes. These results suggest that these closely related antigens can be independently expressed. Analysis of the expression of I and SSEA-1 was then extended to a series of mouse and human cell lines; some express both, some express only one, and some express neither of these antigens. The activation of specific glycosyltransferases and/or glycosidases during development and differentiation appears to be the biochemical mechanism regulating expression of these antigens.     

CRIM1 is involved in endothelial cell capillary formation in vitro and is expressed in blood vessels in vivo

In endothelial cells that form capillary-like structures in vitro a variety of genes is upregulated as we have demonstrated previously. In addition to well known genes, we also identified genes never described in endothelial cells before. Here, we report the further characterization of one selected gene called cysteine-rich motor neuron 1 (CRIM1). CRIM1 is strongly upregulated in endothelial cells during tube formation and is expressed by a variety of adherent growing cell lines whereas cell lines grown in suspension do not express CRIM1. By using antisense technology we were able to inhibit CRIM1 expression and demonstrate impaired formation of capillary-like structures in vitro in transfected endothelial cells. Furthermore, we show that CRIM1 is a glycosylated type I transmembrane protein, that accumulates at sites of close cell-to-cell contact upon stimulation. Finally, we found CRIM1 protein to be expressed by endothelial cells of the inner lining of blood vessels in vivo. Taken together our results imply a possible role of CRIM1 in capillary formation and maintainance during angiogenesis.     

A non-sulfated form of the HNK-1 carbohydrate is expressed in mouse kidney

The HNK-1 carbohydrate, which is recognized by anti-HNK-1 antibody, is well known to be expressed predominantly in the nervous system. The characteristic structural feature of the HNK-1 carbohydrate is 3-sulfo-glucuronyl residues attached to lactosamine structures (Gal beta1-4GlcNAc) on glycoproteins and glycolipids. The biosynthesis of the HNK-1 carbohydrate is regulated mainly by two glucuronyltransferases (GlcAT-P and GlcAT-S) and a sulfotransferase. In this study, we found that GlcAT-S mRNA was expressed at higher levels in the kidney than in the brain, but that both GlcAT-P and HNK-1 sulfotransferase mRNAs, which were expressed at high levels in the brain, were not detected in the kidney. These results suggested that the HNK-1 carbohydrate without sulfate (non-sulfated HNK-1 carbohydrate) is expressed in the kidney. We substantiated this hypothesis using two different monoclonal antibodies: one (anti-HNK-1 antibody) requires sulfate on glucuronyl residues for its binding, and the other (antibody M6749) does not. Western blot analyses of mouse kidney revealed that two major bands (80 and 140 kDa) were detected with antibody M6749, but not with anti-HNK-1 antibody. The 80- and 140-kDa band materials were identified as meprin alpha and CD13/aminopeptidase N, respectively. We also confirmed the presence of the non-sulfated HNK-1 carbohydrate on N-linked oligosaccharides by multistage tandem mass spectrometry. Immunofluorescence staining with antibody M6749 revealed that the non-sulfated HNK-1 carbohydrate was expressed predominantly on the apical membranes of the proximal tubules in the cortex and was also detected in the thin ascending limb in the inner medulla. This is the first study indicating the presence of the non-sulfated HNK-1 carbohydrate being synthesized by GlcAT-S in the kidney. The results presented here constitute novel knowledge concerning the function of the HNK-1 carbohydrate.     

Alpha6beta1 integrin expressed by sperm is determinant in mouse fertilization

Background  

Based on inhibition tests, the alpha6beta1 integrin was suggested to be a sperm receptor, but further experiments using gene deletion techniques have shown that neither oocyte alpha6, nor beta1 integrin subunits were essential for mouse fertilization.     

The neuropeptide-degrading enzyme NL1 is expressed in specific neurons of mouse brain

Metalloendopeptidases of the M13 family were shown to play critical roles in normal physiological processes such as pain control, hypertension and phosphate metabolism, and in pathological states such as Alzheimer's disease. Recently, NL1, a novel member of the family, has been identified and shown to be expressed in several tissues both as a membrane-bound and a secreted protein. As a further step to understand the physiological role(s) of NL1 in mouse, we mapped NL1 mRNA expression pattern in embryos and in young animals at postnatal days p1 and p3, and in adult nervous tissue, using in situ hybridization at the cellular level. No expression could be detected in embryos and young animals. In contrast, NL1 expression was evident in adult brain, pituitary gland and spinal cord. In the central nervous system (CNS), NL1 mRNA was predominantly found in the ventro-posterior regions, which are mostly associated with vegetative functions. At the cellular level, NL1 mRNA was non-uniformly distributed within subpopulations of neurons. In the spinal cord, specific signal was observed in the gray matter. Then, in order to identify putative relevant substrates for NL1, we studied its enzymatic activity towards peptides known to be co-expressed in the NL1-positive domains. Our study showed that NL1 degrades several of these peptides in vitro, the most readily degraded peptides being Bradykinin and Substance P. These results suggest that NL1 is likely to play a critical role in the central nervous system.     

Homeoprotein Hex is expressed in mouse developing chondrocytes

Endochondral ossification is a complex process involving the formation of cartilage and the subsequent replacement by mineralized bone. Although the proliferation and differentiation of chondrocytes are strictly regulated, the molecular mechanisms involved are not completely understood. Here, we show that a divergent-type homeobox gene, hematopoietically expressed homeobox gene (HEX), is expressed in mouse chondrogenic cell line ATDC5. The expression of Hex protein drastically increased during differentiation. The chondrogenic differentiation-enhanced expression of Hex protein was also observed in chondrocytes in the tibia of embryonic day 15.5 (E15.5) mouse embryos. The localization of Hex protein in the chondrocytes of the tibia changed in association with maturation; namely, there was Hex protein in the cytoplasm near the endoplasmic reticulum (ER) in resting chondrocytes, which moved to the nucleus in prehypertrophic chondrocytes, and thereafter entered the ER in hypertrophic chondrocytes. These results suggest Hex expression and subcellular localization are associated with chondrocyte maturation.     

Protocadherin-1 is expressed in the notochord of mouse embryo but is dispensable for its formation

Notochord is an embryonic midline structure that serves as mechanical support for axis elongation and the signaling center for the surrounding tissues. Precursors of notochord are initially induced in the dorsal most mesoderm region in gastrulating embryo and separate from the surrounding mesoderm/endoderm tissue to form an elongated rod-like structure, suggesting that cell adhesion molecules may play an important role in this step. In Xenopus embryo, axial protocadherin (AXPC), an orthologue of mammalian Protocadherin-1 (PCDH1), is indispensable for the assembly and separation from the surrounding tissue of the notochord cells. However, the role of PCDH1 in mammalian notochord remains unknown. We herein report that PCDH1 is expressed in the notochord of mouse embryo and that PCDH1-deficient mice form notochord normally. First, we examined the temporal expression pattern of pcdh1 and found that pcdh1 mRNA was expressed from embryonic day (E) 7.5, prior to the stage when notochord cells detach from the surrounding endoderm tissue. Second, we found that PCDH1 protein is expressed in the notochord of mouse embryos in addition to the previously reported expression in endothelial cells. To further investigate the role of PCDH1 in embryonic development, we generated PCDH1-deficient mice using the CRISPR-Cas9 system. In PCDH1-deficient embryos, notochord formation and separation from the surrounding tissue were normal. Structure and marker gene expression of notochord were also unaffected by loss of PCDH1. Major vascular patterns in PCDH1-deficient embryo were essentially normal. These results suggest that PCDH1 is dispensable for notochord formation, including the tissue separation process, in mammalian embryos. We successfully identified the evolutionary conserved expression of PCDH1 in notochord, but its function may differ among species.     

Staufen1 is expressed in preimplantation mouse embryos and is required for embryonic stem cell differentiation

Pluripotent mouse embryonic stem (mES) cells derived from the blastocyst of the preimplantation embryo can be induced to differentiate in vitro along different cell lineages. However the molecular and cellular factors that signal and/or determine the expression of key genes, and the localisation of the encoded proteins, during the differentiation events are poorly understood. One common mechanism by which proteins can be targeted to specific regions of the cell is through the asymmetric localisation of mRNAs and Staufen, a double-stranded RNA binding protein, is known to play a direct role in mRNA transport and localisation. The aims of the present study were to describe the expression of Staufen in preimplantation embryos and mES cells and to use RNA interference (RNAi) to investigate the roles of Staufen1 in mES cell lineage differentiation. Western blotting and immunocytochemistry demonstrated that Staufen is present in the preimplantation mouse embryo, pluripotent mES cells and mES cells stimulated to differentiate into embryoid bodies, but the Staufen staining patterns did not support asymmetric distribution of the protein. Knockdown of Staufen1 gene expression in differentiating mES cells reduced the synthesis of lineage-specific markers including Brachyury, alpha-fetoprotein (AFP), PAX-6, and Vasa. There was however no significant change in either the gene expression of Nanog and Oct4, or in the synthesis of SSEA-1, all of which are key markers of pluripotency. These data indicate that inhibition of Staufen1 gene expression by RNAi affects an early step in mES cell differentiation and suggest a key role for Staufen in the cell lineage differentiation of mES cells.