Schwann cell-derived desert hedgehog signals nerve sheath formation.

Reciprocal signaling between axons and Schwann cells during development is well established. The contribution of Schwann cells to the formation and maintenance of the protective nerve sheaths (endo-, peri-, and epineurium) has been less studied. Although mesenchymal cells contribute to all these structures, only perineurial cells contribute to the diffusion barrier between nerves and surrounding tissues. During development, prospective perineurial cells shift from a mesenchymal to epithelial phenotype, forming concentric layers of cells around the nerve fascicles that collectively form a barrier against unwanted molecules and cellular infiltration. We have studied the role of Schwann cells in the formation and maintenance of this barrier. The signaling molecule Desert hedgehog is expressed in Schwann cell precursors, and in Schwann cells until at least postnatal day 10, while its receptor patched is seen in mesenchymal cells surrounding the developing nerve at embryo day 15. In Desert hedgehog knockout mice, the connective tissue sheaths in adult nerves appear highly abnormal by electron microscopy. There is almost no epineurium, and the perineurium is thin and highly abnormal. In addition, perineurial-like cells invade the endoneurial space, forming mini-fascicles around small bundles of nerve fibers similar to those seen in regenerating nerves. Functional tests reveal that the diffusion and cellular infiltration barrier is compromised, demonstrating that Desert hedgehog signaling from Schwann cells to the mesenchyme is involved in the formation of a morphologically and functionally normal perineurium.

Schwann cell-derived Desert hedgehog controls the development of peripheral nerve sheaths.

We show that Schwann cell-derived Desert hedgehog (Dhh) signals the formation of the connective tissue sheath around peripheral nerves. mRNAs for dhh and its receptor patched (ptc) are expressed in Schwann cells and perineural mesenchyme, respectively. In dhh-/- mice, epineurial collagen is reduced, while the perineurium is thin and disorganized, has patchy basal lamina, and fails to express connexin 43. Perineurial tight junctions are abnormal and allow the passage of proteins and neutrophils. In nerve fibroblasts, Dhh upregulates ptc and hedgehog-interacting protein (hip). These experiments reveal a novel developmental signaling pathway between glia and mesenchymal connective tissue and demonstrate its molecular identity in peripheral nerve. They also show that Schwann cell-derived signals can act as important regulators of nerve development.

Schwann cell development in embryonic mouse nerves.

Previously we proposed that Schwann cell development from the neural crest is a two-step process that involves the generation of one main intermediate cell type, the Schwann cell precursor. Until now Schwann cell precursors have only been identified in the rat, and much remains to be learned about these cells and how they generate Schwann cells. Here we identify this cell in the mouse and analyze its transition to form Schwann cells in terms of timing, molecular expression, and extracellular signals and intracellular pathways involved in survival, proliferation, and differentiation. In the mouse, the transition from precursors to Schwann cells takes place 2 days earlier than in the rat, i.e., between embryo days 12/13 and 15/16, and is accompanied by the appearance of the 04 antigen and the establishment of an autocrine survival circuit. Beta neuregulins block precursor apoptosis and support Schwann cell generation in vitro, a process that is accelerated by basic fibroblast growth factor 2. The development of Schwann cells from precursors also involves a change in the intracellular survival signals utilized by neuregulins: To block precursor death neuregulins need to signal through both the mitogen-activated protein kinase and the phosphoinositide-3-kinase pathways although neuregulins support Schwann cell survival by signaling through the phosphoinositide-3-kinase pathway alone. Last, we describe the generation of precursor cultures from single 12-day-old embryos, a prerequisite for culture studies of genetically altered precursors when embryos are non-identical with respect to the transgene in question.

Developing Schwann cells acquire the ability to survive without axons by establishing an autocrine circuit involving insulin-like growth factor, neurotrophin-3, and platelet-derived growth factor-BB.

Although Schwann cell precursors from early embryonic nerves die in the absence of axonal signals, Schwann cells in older nerves can survive in the absence of axons in the distal stump of transected nerves. This is crucially important, because successful axonal regrowth in a damaged nerve depends on interactions with living Schwann cells in the denervated distal stump. Here we show that Schwann cells acquire the ability to survive without axons by establishing an autocrine survival loop. This mechanism is absent in precursors. We show that insulin-like growth factor, neurotrophin-3, and platelet-derived growth factor-BB are important components of this autocrine survival signal. The secretion of these factors by Schwann cells has significant implications for cellular communication in developing nerves, in view of their known ability to regulate survival and differentiation of other cells including neurons.

Multiple restricted origin of oligodendrocytes.

The plp gene encodes the proteolipid protein and its alternatively spliced product DM-20, major proteins of CNS myelin. In the mouse, plp/dm-20 transcripts are expressed beginning at embryonic day 9.5 (E9.5) by restricted foci of germinative neuroepithelial cells. To determine the identity of the neural precursors expressing plp/dm- 20, a zeomycin resistance gene fused to the lacZ reporter was expressed in transgenic mice under the control of the plp regulatory sequences. In the three different lines generated, the pattern of beta-galactosidase expression was similar and superimposable on the expression pattern of endogenous plp/dm-20. Both in vivo and in vitro, the transgene was expressed by O4(+) pre-oligodendrocytes, and later by RIP+ differentiated oligodendrocytes, but not by neuronal cells, astrocytes, or radial glial cells. After zeomycin selection, a dramatic enrichment in O4(+) pre-oligodendrocytes was observed in cultures derived from E12.5 transgenic embryos. This enrichment indicates the oligodendroglial specification of neural precursors that continuously express plp/dm-20. Early plp/dm-20-expressing precursors, however, appear to be a separate population from previously described PDGFRalpha oligodendrocyte precursors, as shown by the striking differences in their (1) patterns of distribution and (2) responsiveness to PDGF. These data suggest that oligodendrocytes have a plural origin and that early plp/dm-20 defines one of the neural lineages generating oligodendrocytes.

Identification of transcriptionally regulated mRNAs from mouse Schwann cell precursors using modified RNA fingerprinting methods.

We have adopted RNA fingerprinting methods to screen for genes that are rapidly up- or down-regulated during normal mammalian development, comparing mRNA from early (embryo day 12) to late (embryo day 13) mouse Schwann cell precursors. The use of total RNA, a reduction of cDNA template for amplification, the detection of RT-PCR products with a sensitive DNA stain and polyacrylamide gel electrophoresis and rigid selection criteria involving three screening steps are significant improvements on previous methods. Of 19 differentially displayed bands, 15 represented novel genes. The four known cDNA fragments (interleukin enhancer binding factor 1, beta3 subunit of phospholipase C, brain beta-spectrin, and P21 polypeptide) consisted of coding sequences, indicating a high chance of obtaining coding regions. A semiquantitative RT-PCR analysis of three of the four known genes and a cDNA fragment randomly selected from the pool of 15 novel sequences, confirmed that they were regulated between embryo days 12 and 13, as predicted by the display gels. Our results suggest that the combination of methods described here will have wide applicability in studies of other developmental systems where precisely timed changes occur and where only small amounts of RNA can be obtained for analysis.

PMP-22 expression in the central nervous system of the embryonic mouse defines potential transverse segments and longitudinal columns.

PMP-22, a major constituent of peripheral nervous system (PNS) myelin, is also present in the central nervous system (CNS), in motoneurons of the cranial nerve motor nuclei and spinal cord (Parmantier et al. [1995] Eur. J. Neurosci. 7:1080-1088). The expression of PMP-22 in the CNS during embryonic and early postnatal development was investigated and showed a biphasic spatio-temporal pattern. The expression of PMP-22 started at embryonic day (E)11.5, in restricted longitudinal and transverse domains, in the ventricular zone of the spinal cord, rhombencephalon, mesencephalon and prosencephalon. In the mid- and forebrain, the PMP-22 signal was detectable in a longitudinal domain that followed ventrally the basal/alar boundary but could no longer be detected dorsally at some distance from the roof plate. Along the caudo-rostral axis, the territory in which PMP-22 was detected spanned the mesencephalon and the prosencephalon, extending caudally from the limit between the isthmus and the mesencephalon, and rostrally to the boundary between prosomeres 4 and 5 (p4/p5). In agreement with the prosomeric model of forebrain organization proposed by Puelles and Rubenstein ([1993] TINS 16:472-479), differences in the level of PMP-22 expression in p2, p3, and p4 clearly defined the p2/p3 and p3/p4 neuromeric boundaries. By E17.5, PMP-22 was no longer detected in the ventricular zone, but at E18.5 it began to be expressed in motoneurons of cranial nerve motor nuclei and, after birth, following a rostro-caudal gradient, in the ventral spinal cord.

Oct-6 (SCIP/Tst-1) is expressed in Schwann cell precursors, embryonic Schwann cells, and postnatal myelinating Schwann cells: comparison with Oct-1, Krox-20, and Pax-3.

The POU domain transcription factor Oct-6 (SCIP/Tst-1) is likely to control important stages of Schwann cell development, including the initiation of myelination around birth. Here, we use immunocytochemical and reverse transcriptase-polymerase chain reaction techniques to examine Oct-6 earlier in nerve development, to test the idea that Oct-6 has an additional role in Schwann cell precursors or early embryonic Schwann cells, a possibility raised by previous studies on transgenic mice. Consistent with this, we find low but unambiguous levels of Oct-6 mRNA and protein in Schwann cell precursors of mouse and rat (nerves from 12- and 14-day-old embryos, respectively), with expression levels gradually increasing during early Schwann cell development and towards birth. Unexpectedly, Oct-6 immunoreactivity is clearly present in nuclei of most myelinating cells at least as late as postnatal day 12. Furthermore, many nonmyelinating Schwann cells express Oct-6 in adult life. A comparison of Oct-6 mRNA with other Schwann cell transcription factors-namely, Oct-1, Krox-20, and Pax-3-reveals that each factor exhibits strong developmental regulation and a unique expression pattern in embryonic nerves. Therefore, they are likely to play distinct regulatory roles in early development of the Schwann cell lineage.

Peripheral myelin protein-22 is expressed in rat and mouse brain and spinal cord motoneurons.

Peripheral myelin protein PMP-22 is expressed by Schwann cells and is a constituent of peripheral nervous system (PNS) myelin. Two PMP-22 transcripts, SR13 and CD25, differing in their 5' non-coding sequences have been described. SR13 is present both in the PNS and in non-neural tissue, whereas CD25 mRNA is almost exclusively expressed in Schwann cells. PMP-22 mRNA is also present in the central nervous system (CNS), but at much lower levels than in the PNS. We have investigated the regional distribution of PMP-22 mRNA in the rat and mouse CNS by the reverse transcriptase-polymerase chain reaction method, using oligonucleotide primers specific for the SR13 or CD25 transcripts. SR13 mRNA was detected in all the CNS regions analysed, whereas the CD25 message was present only in the brainstem and the spinal cord. Localization of the PMP-22 transcripts, determined by in situ hybridization in 21 day-old animals, showed selective expression in the motor nuclei. The PMP-22 signal was very weak in the nuclei of the oculomotor and trochlear nerves and absent in the nucleus of the abducens nerve. A strong PMP-22 signal was observed in the motor nuclei of the trigeminal, facial, ambigus, vagus, hypoglossal and accessory spinal nerves and in the ventral horn of the spinal cord. The PMP-22-positive cells were identified as motoneurons on the basis of topographic and morphological criteria, as well as immunolabelling with neuron-specific antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

HIV-1 envelope glycoprotein gp120 does not bind to galactosylceramide-expressing rat oligodendrocytes.

It may be postulated that the encephalopathy induced by the human immunodeficiency virus HIV-1, in particular, the characteristic "myelin pallor," may result from binding of the envelope glycoprotein gp120 to galactosylceramide and/or its metabolite sulfatide in the plasma membrane of oligodendrocytes, the myelin forming cells in the central nervous system. (1) gp120 has been reported to have a high affinity for these molecules in vitro. (2) The binding of antibodies to these molecules increases intracellular free calcium levels, which may be cytotoxic. (3) The binding of gp120 to the CD4 receptor in the immune system has the same effect. We have investigated the binding of gp120 to rat oligodendrocytes in vitro by indirect immunofluorescence and have monitored changes in intracellular free calcium with the calcium-sensitive dye INDO-1, in individual oligodendrocytes exposed to the glycoprotein. Antibodies against galatosylceramide and sulfatide bound to the cell membrane, but gp120 did not. The antibodies also increased intracellular free calcium levels in the oligodendrocytes, whereas gp120 did not. It, therefore, seems highly improbable that the demyelination observed during HIV encephalopathy is a direct cytotoxic effect of gp120 on oligodendrocytes.

The E. coli envY gene encodes a high affinity opioid binding site.

In an attempt to isolate a cDNA encoding an opioid receptor, a cDNA library was constructed in the lambda ZAP vector using NG108-15 mRNA as template. Using an in vitro transcription-translation assay and a sib selection strategy, a single phage was isolated. An RNA transcribed from this cDNA was able to direct in vitro translation of opioid binding sites. The insert was sequenced and comparison with data banks showed a 100% homology with the E. coli envY gene. We assume that the presence of the envY sequence in the NG108-15 cDNA library was due to a contamination of the lambda ZAP vector with E. coli DNA. A search for opioid binding sites on E. coli strains showed that envY+ strains, but not envY- mutants were able to bind opiates. On envY+ cells, the sites are stereospecific, saturable and of high affinity for the opiate ligands. These sites bind opiate agonists and antagonists but neither mu nor delta opioid peptides. In contrast, rabbit reticulocyte lysate primed with RNA transcribed in vitro from the envY sequence elicited the synthesis of an opioid binding site with mixed mu and delta properties. In addition, transfection of the envY sequence into mammalian cells resulted in the expression of opioid binding sites. Depending on the type of cells transfected, these sites were selective for either the mu or delta ligands.

Regional and developmental variations of GFAP and actin mRNA levels in the CNS of jimpy and shiverer mutant mice.

Gliosis is a common reaction to brain damage. Glial fibrillary acidic protein (GFAP) is a classical astrocytic marker. We have undertaken to measure the level of GFAP-mRNA as an index of gliosis in the brain of jimpy (jp) and shiverer (shi) murine mutants, in which hypomyelination is either severe or moderate, respectively. This study was conducted in five different CNS regions and at different ages. In young jp mutant, the amount of GFAP-mRNA was either normal or lower than in control animals; but after 3 wk of age, the level of GFAP-transcript increased dramatically in all regions examined. A parallel increase in actin-mRNA was also observed, mostly in the diencephalon and to a lesser extent in cortex and spinal cord, but not in the cerebellum and brainstem. In the shi mutant, variations in the amount of GFAP-mRNA were less important than in the jp with two exceptions: In brainstem of 3-wk-old animals, a 2.5-fold increase was observed, and in all the regions but the spinal cord of 12-d-old shi, the levels of GFAP-transcript were 2-5 times lower than in controls. In this mutant, the levels of actin message were usually close to normal, or slightly lower than in controls.

Developmental expression of glial fibrillary acidic protein and actin-encoding messages in quaking and control mice.

Quaking is a neurological mutation leading to pleiotropic phenotypic expression, the most prominent being disturbed myelin formation in the central nervous system (CNS) with minor abnormalities in the peripheral nervous system. Previous immunochemical measurements of glial fibrillary acidic protein (GFAP) revealed a marked increase in the protein in several areas of the CNS. To further characterize the regulation parameters of GFAP synthesis, we analyzed the levels of GFAP mRNA in 5 regions of the CNS, some with elevated levels of GFAP and some without. This was compared to the developmental expression of GFAP transcripts in the same regions in normal mice. To establish the specificity of the variations observed with this astroglial specific message, we conducted a similar investigation with actin RNA which is expressed by several cell types in the CNS. Both the actin and the GFAP message were found to be increased in the adult mutant throughout the CNS. In 2-year-old normal mice the messengers for both cytoskeleton proteins were expressed in a higher amount than in young adults.