The adhesion molecule L1 is a member of the immunoglobulin superfamily. L1 is involved in various recognition processes in the CNS and PNS, and binding to L1 can activate signal transduction pathways. Mutations in the human L1 gene are associated with a variable phenotype, including mental retardation and anomalous development of the nervous system, referred to as 'CRASH' (corpus callosum hypoplasia, retardation, adducted thumbs, spastic paraplegia, and hydrocephalus). We generated an animal model of these conditions by gene targetting. Mutant mice were smaller than wild-type and were less sensitive to touch and pain, and their hind-legs appeared weak and uncoordinated. The size of the corticospinal tract was reduced and, depending on genetic background, the lateral ventricles were often enlarged. Non-myelinating Schwann cells formed processes not associated with axons and showed reduced association with axons. In vitro, neurite outgrowth on an L1 substrate and fasciculation were impaired. The mutant mouse described here will help to elucidate the functions of L1 in the nervous system and how these depend on genetic influences.

Jagged1-mediated Notch signaling has been suggested to be critically involved in hematopoietic stem cell (HSC) selfrenewal. Unexpectedly, we report here that inducible Cre-loxP-mediated inactivation of the Jagged1 gene in bone marrow progenitors and/or bone marrow (BM) stromal cells does not impair HSC selfrenewal or differentiation in all blood lineages. Mice with simultaneous inactivation of Jagged1 and Notch1 in the BM compartment survived normally following a 5FU-based in vivo challenge. In addition, Notch1-deficient HSCs were able to reconstitute mice with inactivated Jagged1 in the BM stroma even under competitive conditions. In contrast to earlier reports, these data exclude an essential role for Jagged1-mediated Notch signaling during hematopoiesis. IntroductionHematopoietic stem cells (HSCs) exhibit self-renewing capacity as well as the ability to give rise to more committed progenitors that differentiate into all hematopoietic lineages. 1 The molecular mechanisms regulating stem cell self-renewal and/or differentiation are only poorly understood. Among the proteins that have been postulated to be involved in hematopoietic stem cell maintenance are the Notch receptors and their ligands. 2 Mammals have 4 Notch receptors (Notch1-4) that bind 5 different ligands (Jagged1-2, Delta-like 1-3-4). Expression of a constitutively active form of Notch1 (N1) in murine bone marrow progenitors can lead to increased HSC-self-renewal 3 or to the immortalization of stem cell like progenitors capable of undergoing lymphoid and myeloid differentiation both in vitro and in vivo. 4 In addition, coculture of murine or human HSCs with immobilized Notch ligands, or feeder cells expressing such ligands, can maintain or even enhance HSC self-renewal. [5][6][7][8][9] Recently, osteoblasts expressing the Notch ligand Jagged1 (J1) were identified as being part of the hematopoietic stem cell niche. Osteoblast-specific expression of the activated parathyroid hormonerelated protein receptor results in increased numbers of osteoblasts expressing high levels of J1. The increase in osteoblasts correlates with an increase in the number of HSCs, with evidence of N1 activation in vivo. 10 These results were interpreted to mean that J1-expressing osteoblasts regulate HSC homeostasis through N1 activation.To definitively assess the role of J1 in the hematopoietic system, we have generated inducible gene-targeted mice for J1. Surprisingly, inactivation of J1 in either bone marrow (BM) progenitors or BM stromal cells had no effect on HSC maintenance. In addition, N1-deficient HSCs transplanted into mice with inactivated J1 in the BM stroma reconstituted BM chimeras normally. Our data exclude an essential contribution of J1-mediated N1 signaling for HSC self-renewal or differentiation. Study design Generation and conditional inactivation of mice with a loxP-flanked J1A J1 genomic clone was isolated from a mouse genomic library using an oligonucleotide complementary to part of the first coding exon. LoxP sites were introduced into an XhoI site a...

The demyelinating toxin cuprizone is used increasingly in mouse studies of central nervous system remyelination. The value of this model for such studies depends on an accurate description of its quantifiable features. We therefore investigated histology and ultrastructure during the early oligodendrocyte differentiation phase of remyelination in mice given cuprizone and allowed to recover for 2 weeks. Limiting the dose of cuprizone to 0.2% overcame significant mouse morbidity and weight loss seen with a 0.4% dose, but the distribution of cuprizone‐induced demyelination was anatomically variable. The caudal corpus callosum and dorsal hippocampal commissure mostly demyelinated at this dose, but the rostral corpus callosum and rostral cerebellar peduncles did not. This variable response, together with small axon diameters and hence thin myelin sheaths, hindered analysis of the progress of early remyelination. The proportion of myelinated and unmyelinated axons in defined regions followed expected trends, but there was pronounced variation between animals. Furthermore, group mean G ratios did not change as expected during the early stages of remyelination, and regression analysis revealed a complex relationship between axon diameter and myelin sheath thickness during this period. We also noted axonal pathology that persisted for at least 2 weeks after cuprizone withdrawal.

We report the primary structures of human and rabbit brush border membrane beta‐glycosidase complexes (pre‐pro‐lactase‐phlorizin hydrolase, or pre‐pro‐LPH, EC 3.2.1.23‐62), as deduced from cDNA sequences. The human and rabbit primary translation products contain 1927 and 1926 amino acids respectively. Based on the data, as well as on peptide sequences and further biochemical data, we conclude that the proteins comprise five domains: (i) a cleaved signal sequence of 19 amino acids; (ii) a large ‘pro’ portion of 847 amino acids (rabbit), none of which appears in mature, membrane‐bound LPH; (iii) the mature LPH, which contains both the lactase and phlorizin hydrolase activities in a single polypeptide chain; (iv) a membrane‐spanning hydrophobic segment near the carboxy terminus, which serves as membrane anchor; and (v) a short hydrophilic segment at the carboxy terminus, which must be cytosolic (i.e. the protein has an Nout‐Cin orientation). The genes have a 4‐fold internal homology, suggesting that they evolved by two cycles of partial gene duplication. This repetition also implies that parts of the ‘pro’ portion are very similar to parts of mature LPH, and hence that the ‘pro’ portion may be a water‐soluble beta‐glycosidase with another cellular location than LPH. Our results have implications for the decline of LPH after weaning and for human adult‐type alactasia, and for the evolutionary history of LPH.

We have selectively inhibited Notch1 signaling in oligodendrocyte precursors (OPCs) using the Cre/loxP system in transgenic mice to investigate the role of Notch1 in oligodendrocyte (OL) development and differentiation. Early development of OPCs appeared normal in the spinal cord. However, at embryonic day 17.5, premature OL differentiation was observed and ectopic immature OLs were present in the gray matter. At birth, OL apoptosis was strongly increased in Notch1 mutant animals. Premature OL differentiation was also observed in the cerebrum, indicating that Notch1 is required for the correct spatial and temporal regulation of OL differentiation in various regions of the central nervous system. These findings establish a widespread function of Notch1 in the late steps of mammalian OPC development in vivo.

The 12 interferon (IFN)-related sequences detected in a human gene bank fall into not less than eight distinct classes, indicating that there are at least eight IFN-related genes. Most, if not all, of these direct the synthesis of an IFN in Escherichia coli. The sequence of one chromosomal gene and its flanking regions was identical to that deduced for the cDNA corresponding to IFN-alpha l mRNA. No evidence was found for the existence of an intron, in either the coding or the non-coding segments of the gene.

A cDNA library from rabbit kidney cortex was screened for expression of Na-dependent transport of phosphate (P.) using Xenopus laevis oocytes as an expression system. A single clone was eventually isolated (designated NaPi-1) that stimulated expression of Na/P1 cotransport -700-fold compared to total mRNA. The predicted sequence of the Na/P1 cotransporter consists of 465 amino acids (relative molecular mass, 51,797); hydropathy profile predictions suggest six (possibly eight) membrane-spanning segments. In vitro translation of NaPi-1/complementary RNA in the presence of pancreatic microsomes indicated NaPi-1 to be a glycosylated protein; four potential N-glycosylation sites are present in the amino acid sequence. Northern blot analysis demonstrated the presence of NaPi-1/mRNA in kidney cortex and liver; no hybridization signal was obtained with mRNA from other tissues (including small intestine). Kinetic analysis of Na/P1 cotransport expressed by NaPi-1/complementary RNA demonstrated characteristics (sodium interaction) similar to those observed in cortical apical membranes. The alignment of 5 amino acid residues (Gly32/Alae'-Xaa-Xaa-Xaa-Xaa-Leu38-Xaa-XaaXaa-Pro3W.Arg39l) is consistent with a motif proposed for Na-dependent transport systems. We conclude that we have cloned a cDNA for a Na/P1 cotransport system present in rabbit kidney cortex.Reabsorption of phosphate (P) in the proximal tubule of the kidney contributes essentially to maintenance of the body Pi homeostasis (1). Influx of Pi at the brush border membrane of epithelial cells is mediated by a Na/Pt cotransporter and is driven by the transmembrane electrochemical potential gradient of sodium (2). Thereafter, P1 moves to the blood across the basolateral membrane, most likely via an anion-exchange mechanism and/or another Na-dependent Pi transport system. This transepithelial transport of P1 is controlled in a complex manner by various hormonal (e.g., parathyroid hormone) and nonhormonal (e.g., dietary Pi/Pi demand) factors (2-4).By using different experimental systems such as isolated tubules, isolated brush border membranes, and established cell cultures, it has been demonstrated that regulation of proximal tubular Pi reabsorption is accomplished mainly by modulation of the apically localized Na/Pi cotransport system (2). Thus, this Na/Pa cotransport system is a central target within the complex control of P1 homeostasis. Studies with established cell lines (mainly opossum kidney cells) demonstrated that inhibition of the Na/Ps cotransport (by, for example, parathyroid hormone) is mediated by activation of protein kinase C and/or A followed by an internalization step (endocytosis) of the transport system. On the other hand, stimulation of Na/P, cotransport by, for example, reduction of the concentration of extracellular Pi has been demonstrated to be dependent on de novo protein synthesis (2, 5).Despite the detailed knowledge of kinetic and regulatory properties of the renal (proximal tubular) Na/Pa cotransport system and despite several bioc...

Neural stem cells (NSCs) in the postnatal mammalian brain self-renew and are a source of neurons and glia. To date, little is known about the molecular and cellular mechanisms regulating the maintenance and differentiation of these multipotent progenitors. We show that Jagged1 is required by mitotic cells in the subventricular zone (SVZ) and stimulates self-renewal of multipotent epidermal growth factor-dependent NSCs. Jagged1-expressing cells line the adult SVZ and are juxtaposed to Notch1-expressing cells, some of which are putative NSCs. In vitro, endogenous Jagged1 acts through Notch1 to promote NSC maintenance and multipotency. In vivo, reducing Jagged1/Notch1 signaling decreases the number of proliferating cells in the SVZ. In addition, soluble Jagged1 promotes self-renewal and neurogenic potential of multipotent neural progenitors in vitro. Our findings suggest a central role for Jagged1 in the NSC niche in the SVZ for maintaining a population of NSCs in the postnatal brain.