Importing genetically altered animals: ensuring quality.

The reproducibility of research using laboratory animals requires reliable management of their quality, in particular of their genetics, health and environment, all of which contribute to their phenotypes. The point at which these biological materials are transferred between researchers is particularly sensitive, as it may result in a loss of integrity of the animals and/or their documentation. Here, we describe the various aspects of laboratory animal quality that should be confirmed when sharing rodent research models. We also discuss how repositories of biological materials support the scientific community to ensure the continuity of the quality of laboratory animals. Both the concept of quality and the role of repositories themselves extend to all exchanges of biological materials and all networks that support the sharing of these reagents.

Ptchd1 deficiency induces excitatory synaptic and cognitive dysfunctions in mouse.

Synapse development and neuronal activity represent fundamental processes for the establishment of cognitive function. Structural organization as well as signalling pathways from receptor stimulation to gene expression regulation are mediated by synaptic activity and misregulated in neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability (ID). Deleterious mutations in the PTCHD1 (Patched domain containing 1) gene have been described in male patients with X-linked ID and/or ASD. The structure of PTCHD1 protein is similar to the Patched (PTCH1) receptor; however, the cellular mechanisms and pathways associated with PTCHD1 in the developing brain are poorly determined. Here we show that PTCHD1 displays a C-terminal PDZ-binding motif that binds to the postsynaptic proteins PSD95 and SAP102. We also report that PTCHD1 is unable to rescue the canonical sonic hedgehog (SHH) pathway in cells depleted of PTCH1, suggesting that both proteins are involved in distinct cellular signalling pathways. We find that Ptchd1 deficiency in male mice (Ptchd1-/y) induces global changes in synaptic gene expression, affects the expression of the immediate-early expression genes Egr1 and Npas4 and finally impairs excitatory synaptic structure and neuronal excitatory activity in the hippocampus, leading to cognitive dysfunction, motor disabilities and hyperactivity. Thus our results support that PTCHD1 deficiency induces a neurodevelopmental disorder causing excitatory synaptic dysfunction.

Reg-2 is a motoneuron neurotrophic factor and a signalling intermediate in the CNTF survival pathway.

Cytokines that are related to ciliary neurotrophic factor (CNTF) are physiologically important survival factors for motoneurons, but the mechanisms by which they prevent neuronal cell death remain unknown. Reg-2/PAP I (pancreatitis-associated protein I), referred to here as Reg-2, is a secreted protein whose expression in motoneurons during development is dependent on cytokines. Here we show that CNTF-related cytokines induce Reg-2 expression in cultured motoneurons. Purified Reg-2 can itself act as an autocrine/paracrine neurotrophic factor for a subpopulation of motoneurons, by stimulating a survival pathway involving phosphatidylinositol-3-kinase, Akt kinase and NF-kappaB. Blocking Reg-2 expression in motoneurons using Reg-2 antisense adenovirus specifically abrogates the survival effect of CNTF on cultured motoneurons, indicating that Reg-2 expression is a necessary step in the CNTF survival pathway. Reg-2 shows a unique pattern of expression in late embryonic spinal cord: it is progressively upregulated in individual motoneurons on a cell-by-cell basis, indicating that only a fraction of motoneurons in a given motor pool may be exposed to cytokines. Thus, Reg-2 is a neurotrophic factor for motoneurons, and is itself an obligatory intermediate in the survival signalling pathway of CNTF-related cytokines.

A novel rat tetraspan protein in cells of the oligodendrocyte lineage.

The tetraspanin/transmembrane 4 superfamily gene superfamily encodes proteins that span the plasma membrane four times. Tetraspan proteins are implicated in proliferation, motility, and differentiation in various cell types, and in some cells they may link plasma membrane proteins into signalling complexes. Using a subtractive cDNA library prepared from oligodendrocytes and their progenitor cells, we have identified Tspan-2 as a member of this superfamily. In situ hybridization analysis revealed robust expression in cells of the oligodendrocyte lineage in comparison with the Plp gene, a well-characterized marker for myelin-forming glia in the CNS. Rat Tspan-2 mRNA is restricted to the nervous system and is detectable by northern blot shortly after birth in the CNS. Subsequently the gene is up-regulated strongly between postnatal day 3 and 10, and expression levels continue to rise up to postnatal day 22. These data indicate that Tspan-2 is likely to play a role in signalling in oligodendrocytes in the early stages of their terminal differentiation into myelin-forming glia and may also function in stabilizing the mature sheath.

A study of the potential of the embryonic rat telencephalon to generate oligodendrocytes.

A major question in neural development is whether each part of the telencephalon has an equal potential to generate each cell type. In this study, we address this question specifically in regard to the generation of oligodendrocytes. We cultured precursor cells from two different regions of the rat embryonic telencephalon--the ganglionic eminence that the anlage of the cerebral cortex--from different stages of development, and labeled the cells with a retroviral vector to follow their fate. We discovered that multipotential precursor cells from E13 ganglionic eminence have several orders of magnitude higher capacity to generate oligodendrocyte than the equivalent cells from E13 cerebral cortex. This failure of cortical precursor cells to generate oligodendrocytes at early development stages (E12-E13) could not be reversed by growth factors, permissive growth media, or a permissive striatal cell environment. A combination of striatal contact and plus specific growth factors, however, did induce the production of oligodendrocytes. We conclude that telencephalic precursor cells do not have the potential to generate oligodendrocytes, but that this potential is significantly greater in striatal than cortical multipotential precursor cells.

The regulation of the expression, phosphorylation, and protein associations of pp125FAK during rat brain development.

We have studied both the expression and the interactions of focal adhesion kinase (FAK) during brain development. We have discovered that during different periods of development, FAK apparently has different properties. During the early stage of neurogenesis, FAK is phosphorylated, shows multiple isoforms, and interacts with the proto-oncogenes, src, fyn, and lyn. At this stage, FAK also interacts with both the N- and C-terminal SH2 domains of GAP, a negative regulator of the ras pathway. During later embryonic development, none of these protein interactions are apparent even though FAK is still predominantly phosphorylated. By adulthood FAK is largely unphosphorylated and migrates as a single protein species on SDS--PAGE. We discuss these results in terms of the dynamic cell movements that occur during embryonic brain development.

Influence of growth factors on neuronal differentiation.

With so many neurotrophins and receptors now known, how is our picture of neurotrophism changing? Recent studies on knockout mice have confirmed our expectations of neurotrophin action in neuronal development. A notable exception is the activation of TrkB, on motor neurons, by an unknown ligand. It is also clear that some neurotrophins have diverse activities and influence early developmental stages. There are interesting new data concerning the role of p75, the low affinity neurotrophin receptor, as a modulator of neurotrophin activity. Even more exciting are new studies on glia-derived neurotrophic factor (GDNF) which demonstrate that this growth factor acts as a potential protector of motor neurons and striatal dopaminergic neurons.

Developmental expression of the C1G5F2 antigen in cultured rat oligodendroglial cells.

The C1G5F2 antigen is a newly described minor myelin antigen of the central nervous system. Its expression compared with that of some other main myelin protein components (Wolfgram W1 protein, myelin basic proteins (MBP) and proteolipids) was investigated in rat oligodendrocytes derived from 10-day-old primary glial cell cultures and subcultured for several days in a chemically defined medium. It was demonstrated immunocytochemically that this antigen is detected later than the major myelin markers. All cells immunoreactive with the monoclonal antibody C1G5F2 were always labeled either by W1-, MBP- or proteolipid-specific antisera. It was also shown at the electron microscopic level that this antigen is mainly expressed on the surface of the extremities of the fine oligodendroglial processes. All these observations suggest that the C1G5F2 antigen may be a useful marker for a specific step in the oligodendrocyte maturation stage.

Comparative immunohistochemical study of C1G5F2 antigen and myelin/oligodendrocyte glycoprotein (MOG) expression in brain of several animal species.

The localisation of the myelin/oligodendrocyte specific antigen C1G5F2 and the myelin/oligodendrocyte glycoprotein (MOG) was studied in parallel in the brain of various species throughout the phylogenetic line of vertebrates. This immunofluorescence study was performed on unfixed brain sections by using the newly described monoclonal antibody C1G5F2 and polyclonal anti-MOG antibody. The antigen C1G5F2 is detected from the reptilian class onwards whereas MOG is only found in mammals. Both antibodies clearly stained only the myelin sheaths in the brain of adult animals. The phylogenetic distribution of the C1G5F2 antigen compared to the other well known myelin proteins may indicate that it has a specific function during myelination. Moreover, evidence is given that the C1G5F2 antigen is a new minor myelin protein distinct from the glycoprotein MOG.

A new oligodendrocyte specific plasma membrane surface protein identified by a monoclonal antibody produced in vitro.

This paper describes a novel monoclonal antibody (C1G5F2) derived from mice splenocytes immunized in vitro with a wheat germ agglutinin glycoprotein fraction isolated from bovine central nervous system (CNS) myelin. Immunohistochemical reactions with C1G5F2 were investigated on rat brain sections during the active period of myelination. From day 10 to 13 postnatally, no stained structures were observed throughout the whole brain. The first immunolabeled myelin fibers were detected within the pons at day 14, and the white matter areas in the cerebrum started to be stained some days later. White matter areas of the cerebellum were clearly immunopositive after the third week. There was a strong positive signal on myelin fibers in the cerebrum at day 30. By contrast, no immunolabeled cell bodies of oligodendrocytes were observed throughout the brain. The other neural cell types were also not labeled. This C1G5F2 monoclonal antibody bound mainly to the extracytosolic membrane surface of the processes of live cultured oligodendrocytes derived from newborn rat brain but was unreactive with live or fixed astrocytes and neurons maintained in culture. No immunostaining was detected in the peripheral nervous system or in the spleen, liver, or pancreas. The C1G5F2 epitope containing antigen may therefore be considered as a CNS myelin/oligodendrocyte specific molecule. Sodium deoxycholate-Tween 20 extracts of secondary oligodendrocyte cultures, biotinylated with biotin hydrazide, were used to attempt the purification of the antigen with C1G5F2 IgMs linked to antimouse IgM agarose. A main broad biotinylated protein band of 54-58 kDa molecular mass was noted. In a second approach, the antigen was immunopurified from cultured oligodendrocytes as an immune complex using biotinylated C1G5F2 IgMs. A distinct protein doublet of 53-56 kDa was also observed. It is postulated that this antigen may play an essential role in myelin formation and could be a possible target in diseases restricted to CNS myelin.

Biochemical and immunohistochemical studies with specific polyclonal antibodies directed against bovine myelin/oligodendrocyte glycoprotein.

Bovine myelin/oligodendrocyte glycoprotein (MOG) was purified from a Wolfgram protein fraction of brain myelin by molecular sieving and preparative gel electrophoresis. The N-terminal sequence of this wheat germ agglutinin reacting glycoprotein was determined. Antibodies against purified MOG and synthetic N-terminal octapeptide of MOG were produced in rabbits. Respective affinity purified antibody preparations gave identical results on Western blots. Treatment with specific glycosidases indicated that the oligosaccharide chains of MOG are only of N-chain type. This glycoprotein seems to be restricted to mammalian species since it was not detected in other animal species, ranging from fish up to reptiles. Immunohistochemical investigations on rat brain sections revealed that MOG is restricted to myelin sheaths and oligodendrocytes, thus corroborating previous results obtained with the MOG 8-18C5 monoclonal antibody. Decreased staining pattern in Jimpy brain further attested its specific localization in myelin-related structures. The octapeptide site-specific antibodies were not reactive on brain sections which may be attributed to the burying of this N-terminal sequence in the membrane. These MOG polyclonal antibodies appear to be valuable tools for further studies concerning this minor glycoprotein.

Proteolipid DM-20 predominates over PLP in peripheral nervous system.

The major central nervous system (CNS) myelin proteolipid (PLP) is also expressed in the peripheral nervous system (PNS). This paper gives evidence that DM-20, an isoform of PLP, also occurs in rat sciatic nerves, where, in contrast to what is seen in CNS myelin, it predominates over PLP. This conclusion was reached on the basis of results obtained by immunoblot analysis of a crude proteolipid extract from adult peripheral nerve with two site-specific anti-proteolipid (PLP and DM-20) antibodies. This finding was further corroborated by characterization of the products obtained by Polymerase Chain Reaction (PCR) amplification of cDNAs synthesized from total RNA of 14-day-old sciatic nerves. The significance of the occurrence of these proteolipids in PNS remains obscure.