The subventricular zone: new molecular and cellular developments.

The subventricular zone (SVZ), which lines the lateral walls of the lateral ventricle, persists as a neurogenic zone into adulthood and functions as the largest site of neurogenesis in the adult brain. In recent years, with the acceptance of the concept of postembryonic mammalian neurogenesis, neurogenesis in the adult SVZ has been an area of active research. With the rapid accumulation of new information on the SVZ, some of which is contradictory, summarizing existing knowledge on the SVZ and outlining future research directions in this area become important. In this review, we will cover recent molecular and cellular investigations that characterize the SVZ niche, SVZ neurogenesis, and SVZ cell migration within the adult brain.

Use of coisogenic host blastocysts for efficient establishment of germline chimeras with C57BL/6J ES cell lines.

Gene targeting in embryonic stem (ES) cells allows the production of mice with specified genetic mutations. Currently, germline-competent ES cell lines are available from only a limited number of mouse strains, and inappropriate ES cell/host blastocyst combinations often restrict the efficient production of gene-targeted mice. Here, we describe the derivation of C57BL/6J (B6) ES lines and compare the effectiveness of two host blastocyst donors, FVB/NJ (FVB) and the coisogenic strain C57BL/6-Tyr(c)-2J (c2J), for the production of germline chimeras. We found that when B6 ES cells were injected into c2J host blastocysts, a high rate of coat-color chimerism was detected, and germline transmission could be obtained with few blastocyst injections. In all but one case, highly chimeric mice transmitted to 100% of their offspring. The injection of B6 ES cells into FVB blastocysts produced some chimeric mice. However; the proportion of coat-color chimerism was low, with many more blastocyst injections required to generate chimeras capable of germline transmission. Our data support the use of the coisogenic albino host strain, c2J, for the generation of germline-competent chimeric mice when using B6 ES cells.

Disruption of Eph/ephrin signaling affects migration and proliferation in the adult subventricular zone.

The subventricular zone (SVZ) of the lateral ventricles, the largest remaining germinal zone of the adult mammalian brain, contains an extensive network of neuroblasts migrating rostrally to the olfactory bulb. Little is known about the endogenous proliferation signals for SVZ neural stem cells or guidance cues along the migration pathway. Here we show that the receptor tyrosine kinases EphB1-3 and EphA4 and their transmembrane ligands, ephrins-B2/3, are expressed by cells of the SVZ. Electron microscopy revealed ephrin-B ligands associated with SVZ astrocytes, which function as stem cells in this germinal zone. A three-day infusion of the ectodomain of either EphB2 or ephrin-B2 into the lateral ventricle disrupted migration of neuroblasts and increased cell proliferation. These results suggest that Eph/ephrin signaling is involved in the migration of neuroblasts in the adult SVZ and in either direct or indirect regulation of cell proliferation.

Neurotrophin regulation of the developing nervous system: analyses of knockout mice.

The neurotrophins, NGF, BDNF, NT3 and NT4, are one family in a growing repertoire of neurotrophic factors. The neurotrophins have long been implicated in neuronal survival and recent studies from mice with targeted disruptions of the neurotrophin genes confirm this role, but also reveal that the action of the neurotrophins is more complex, and in some instances more interactive, than originally envisaged. Lack of functional NGF, BDNF and NT3 genes results in severe neuronal deficits and an early postnatal death. However, NT4 is unique among the neurotrophins and while the absence of NT4 does result in limited sensory neuron loss these mice do not die early, suggesting that NT4-dependent neurons are not critical for survival. Phenotypic analyses of mice lacking neurotrophin receptors, TrkA, B and C, confirm that TrkA is the functional receptor for NGF, TrkB acts as the primary receptor for BDNF and NT4, and NT3 signals primarily through TrkC. However, the finding that TrkC mutant mice have a less dramatic phenotype than their NT3 counterparts implicates NT3 in signaling via receptors other than TrkC. Further studies, using combinatorial Trk and neurotrophin deletions, reveal that while BDNF and NT4 subserve distinct neuron populations in most cases, other neuron sub-populations can be supported by either BDNF or NT4, providing evidence for compensatory actions between neurotrophins. As a mechanism to explain programmed cell death that occurs in the developing nervous system, recent studies examining neurotrophin gene-dosage effects suggest that the availability of neurotrophins, NGF, BDNF and NT3, may be limiting for some neuron populations. In addition, the proposed switch in neurotrophin dependency for some neuron populations is now being determined using neurotrophin mutant mice. We discuss these and other recent findings on neurotrophin requirements for the developing nervous system.

Mice lacking brain-derived neurotrophic factor exhibit visceral sensory neuron losses distinct from mice lacking NT4 and display a severe developmental deficit in control of breathing.

The neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT4) act via the TrkB receptor and support survival of primary somatic and visceral sensory neurons. The major visceral sensory population, the nodose-petrosal ganglion complex (NPG), requires BDNF and NT4 for survival of a full complement of neurons, providing a unique opportunity to compare gene dosage effects between the two TrkB ligands and to explore the possibility that one ligand can compensate for loss of the other. Analysis of newborn transgenic mice lacking BDNF or NT4, or BDNF and NT4, revealed that survival of many NPG afferents is proportional to the number of functional BDNF alleles, whereas only one functional NT4 allele is required to support survival of all NT4-dependent neurons. In addition, subpopulation analysis revealed that BDNF and NT4 can compensate for the loss of the other to support a subset of dopaminergic ganglion cells. Together, these data demonstrate that the pattern of neuronal dependencies on BDNF and NT4 in vivo is far more heterogeneous than predicted from previous studies in culture. Moreover, BDNF knockout animals lack a subset of afferents involved in ventilatory control and exhibit severe respiratory abnormalities characterized by depressed and irregular breathing and reduced chemosensory drive. BDNF is therefore required for expression of normal respiratory behavior in newborn animals.

Degeneration of vestibular neurons in late embryogenesis of both heterozygous and homozygous BDNF null mutant mice.

The generation of mice lacking specific neurotrophins permits evaluation of the trophic requirements of particular neuronal populations throughout development. In the present study, we examined the developing vestibulocochlear system to determine the time course of neurotrophin dependence and to determine whether competition occurred among developing cochlear or vestibular neurons for available amounts of either brain-derived neurotrophic factor (BDNF) or neurotrophin-4/5 (NT-4/5). Both cochlear and vestibular neurons were present in mice lacking NT-4/5. In contrast, vestibular neurons decreased in number beginning at mid-stages of inner ear development, in mice lacking BDNF. Early in development (E12.5-13), the size of the vestibular ganglion was normal in bdnf -/- mice. Decreased innervation to vestibular sensory epithelia was detected at E13.5-15, when progressive loss of all afferent innervation to the semicircular canals and reduced innervation to the utricle and saccule were observed. At E16.5-17, there was a reduction in the number of vestibular neurons in bdnf -/- mice. A further decrease in vestibular neurons was observed at P1 and P15. Compared to bdnf -/- mice, mice heterozygous for the BDNF null mutation (bdnf +/-) showed an intermediate decrease in the number of vestibular neurons from E16.5-P15. These data indicate a late developmental requirement of vestibular neurons for BDNF and suggest competition among these neurons for limited supplies of this factor.

Mice lacking the CNTF receptor, unlike mice lacking CNTF, exhibit profound motor neuron deficits at birth.

Ciliary neurotrophic factor (CNTF) supports motor neuron survival in vitro and in mouse models of motor neuron degeneration and was considered a candidate for the muscle-derived neurotrophic activity that regulates motor neuron survival during development. However, CNTF expression is very low in the embryo, and CNTF gene mutations in mice or human do not result in notable abnormalities of the developing nervous system. We have generated and directly compared mice containing null mutations in the genes encoding CNTF or its receptor (CNTFR alpha). Unlike mice lacking CNTF, mice lacking CNTFR alpha die perinatally and display severe motor neuron deficits. Thus, CNTFR alpha is critical for the developing nervous system, most likely by serving as a receptor for a second, developmentally important, CNTF-like ligand.

Neuronal deficits, not involving motor neurons, in mice lacking BDNF and/or NT4.

Nerve growth factor and other neurotrophins signal to neurons through the Trk family of receptor tyrosine kinases. TrkB is relatively promiscuous in vitro, acting as a receptor for brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT4) and, to a lesser extent, NT3 (refs 3-5). Mice lacking TrkB show a more severe phenotype than mice lacking BDNF, suggesting that TrkB may act as a receptor for additional ligands in vivo. To explore this possibility, we generated mice lacking NT4 or BDNF as well as mice lacking both neurotrophins. Unlike mice lacking other Trks or neurotrophins, NT4-deficient mice are long-lived and show no obvious neurological defects. Analysis of mutant phenotypes revealed distinct neuronal populations with different neurotrophin requirements. Thus vestibular and trigeminal sensory neurons require BDNF but not NT4, whereas nodose-petrosal sensory neurons require both BDNF and NT4. Motor neurons, whose numbers are drastically reduced in mice lacking TrkB, are not affected even in mice lacking both BDNF and NT4. These results suggest that another ligand, perhaps NT3, does indeed act on TrkB in vivo.

A BDNF autocrine loop in adult sensory neurons prevents cell death.

During the initial phase of their development, sensory neurons of the dorsal root ganglion (DRG) require target-derived trophic support for their survival, but as they mature they lose this requirement. Because many of these neurons express BDNF (brain-derived neurotrophic factor) messenger RNA, we hypothesized that BDNF might act as an autocrine survival factor in adult DRG neurons, thus explaining their lack of dependence on exogenous growth factors. When cultured adult DRG cells were treated with antisense oligonucleotides to BDNF, expression of BDNF protein was reduced by 80%, and neuronal survival was reduced by 35%. These neurons could be rescued by exogenous BDNF or neurotrophin-3, but not by other growth factors. Similar results were obtained with single-neuron microcultures, whereas microcultures derived from mutant mice lacking BDNF were unaffected by antisense oligonucleotides. Our results strongly support an autocrine role for BDNF in mediating the survival of a subpopulation of adult DRG neurons.

Ciliary neurotrophic factor maintains the pluripotentiality of embryonic stem cells.

Ciliary neurotrophic factor was discovered based on its ability to support the survival of ciliary neurons, and is now known to act on a variety of neuronal and glial populations. Two distant relatives of ciliary neurotrophic factor, leukemia inhibitory factor and oncostatin M, mimic ciliary neurotrophic factor with respect to its actions on cells of the nervous system. In contrast to ciliary neurotrophic factor, leukemia inhibitory factor and oncostatin M also display a broad array of actions on cells outside of the nervous system. The overlapping activities of leukemia inhibitory factor, oncostatin M and ciliary neurotrophic factor can be attributed to shared receptor components. The specificity of ciliary neurotrophic factor for cells of the nervous system results from the restricted expression of the alpha component of the ciliary neurotrophic factor receptor complex, which is required to convert a functional leukemia inhibitory factor/oncostatin M receptor complex into a ciliary neurotrophic factor receptor complex. The recent observation that the alpha component of the ciliary neurotrophic factor receptor complex is expressed by very early neuronal precursors suggested that ciliary neurotrophic factor may act on even earlier precursors, particularly on cells previously thought to be targets for leukemia inhibitory factor action. Here we show the first example of ciliary neurotrophic factor responsiveness in cells residing outside of the nervous system by demonstrating that embryonic stem cells express a functional ciliary neurotrophic factor receptor complex, and that ciliary neurotrophic factor is similar to leukemia inhibitory factor in its ability to maintain the pluripotentiality of these cells.

Gene sequences of chicken BDNF and NT-3.

The respective amino acid sequences of mature brain-derived neurotrophic factor (BDNF) and of mature neurotrophin-3 (NT-3) are identical among mammals, making these among the structurally conserved factors known. Here we show that only a single conservative amino acid substitution distinguishes the chicken mature NT-3 protein from its mammalian counterpart. Chicken mature BDNF shows slightly more variation, differing from mammalian BDNF at several positions. We also note the presence of amino acid sequence motifs in the precursor protein sequences of chicken BDNF and NT-3 that are universally conserved among all known mammalian neurotrophin precursors and have been demonstrated to play a crucial role in promoting correct processing of the pro-proteins.

Stage-specific expression of a family of proteins that are major products of zygotic gene activation in the mouse embryo.

Transcriptional activation of the embryonic genome occurs during the two-cell stage in the mouse embryo and is marked by the synthesis of a set of alpha-amanitin-sensitive proteins of Mr 73,000, 70,000, and 68,000. We have characterized these three proteins by two-dimensional gel electrophoresis of [35S]methionine radiolabeled two-cell embryos. Their isoelectric points range from 6.2 to 6.8 and their synthesis, which can constitute 5-10% of total protein synthesis, is restricted to the two-cell stage. These proteins are not heat shock proteins that have previously been reported as major products of transcriptional activation. Peptide mapping by limited proteolysis indicates that these three proteins are highly related to one another and the results of pulse-chase experiments indicate that they are likely to be degraded by the eight-cell stage. These proteins are nuclear-associated and insoluble in 2% Triton X-100/0.3 M KCl. Although these proteins share some features with somatic lamins--they exhibit solubility properties similar to somatic lamins--they do not cross-react with polyclonal antibodies to either lamins A/C or B, nor do they comigrate with somatic lamins on two-dimensional gels. Additional evidence that these proteins are not lamins is that although treatment of two-cell embryos with okadaic acid, which is an inhibitor of protein phosphatases 1 and 2A, results in precocious nuclear envelope breakdown, the proteins remain insoluble in 2% Triton X-100/0.3 M KCl.

Regulation of mouse preimplantation development: differential effects of CZB medium and Whitten's medium on rates and patterns of protein synthesis in 2-cell embryos.

In contrast to Whitten's medium, CZB medium, which lacks glucose but is supplemented with glutamine, supports development of 1-cell embryos beyond the 2-cell stage for embryos obtained from mice that exhibit the 2-cell block. The molecular basis for this effect, however, is not known. We report that correlated with the ability of CZB medium to support development beyond the 2-cell stage is an enhanced rate of total protein synthesis and higher level of synthesis of proteins that reflect activation of transcription of the embryonic genome when compared to embryos cultured in Whitten's medium +/- ethylenediaminetetraacetate (EDTA). The overall patterns of protein synthesis of embryos cultured in CZB or Whitten's medium +/- EDTA are similar, although the synthesis of the transcription-dependent proteins is markedly delayed in embryos cultured in Whitten's-EDTA. Last, there are no significant differences in the adenosine 5'-triphosphate (ATP) levels in 2-cell embryos cultured from the 1-cell stage in each of the media.

Failure of spermatozoa from T/t mice to fertilize in vitro is overcome by zona drilling.

Failure of epididymal spermatozoa from T/t mutant mice, but not from t/t individuals, to fertilize oocytes in vitro was partially overcome by opening a small aperture in the zona pellucida with acidified Tyrode's solution to permit direct access of the spermatozoon to the vitellus. This study provides a model system to evaluate requirements for successful zona drilling in the treatment of human infertility and further insights into the effects of the t complex on sperm fertility.

Failed fertilization in human in vitro fertilization analyzed with the deoxyribonucleic acid-specific fluorochrome Hoechst 33342.

The degree and normality of nuclear maturation were assessed with the fluorochrome Hoechst 33342 in two groups of inseminated human oocytes that had failed to undergo fertilization. Group 1 consisted of 67 oocytes from 27 patients, each of whom had at least two other oocytes that had been fertilized and had cleaved. Group 2 consisted of 65 oocytes from 14 patients, none of whose oocytes had been fertilized. In group 1, 52.3% of the oocytes were found to be immature (germinal vesicle stage or metaphase-telophase I), whereas in group 2 only 26% were found to be immature. Thus oocyte nuclear immaturity was the major cause of fertilization failure when companion oocytes were fertilized. When no oocytes of a patient were fertilized, most oocytes were found to be mature, so other factors, such as sperm dysfunction or zona binding abnormalities, must account for most of the fertilization failure in this group of patients.

Fertilization of zona-drilled mouse oocytes treated with a monoclonal antibody to the zona glycoprotein, ZP3.

Opening a small aperture in the zona pellucida of mouse oocytes by using micromanipulation and a stream of acidified Tyrode's solution (zona drilling) improved the efficiency of in vitro fertilization at low sperm concentrations without adversely affecting development to the blastocyst stage. Zona drilling also permitted in vitro fertilization and development when sperm penetration through the zona was blocked by a monoclonal antibody to the protein core of the zona glycoprotein, ZP3. These results provide a direct demonstration that sperm entry occurs through the aperture and also suggest that zona drilling of human oocytes may offer a therapeutic approach when autoantibodies to the zona pellucida are suspected as a cause of infertility.

Pre-loading of mouse oocytes with DNA-specific fluorochrome (Hoechst 33342) permits rapid detection of sperm-oocyte fusion.

Mouse oocytes exposed to 1 microgram Hoechst 33342 (H-33342)/ml and then fertilized in vitro developed normally into blastocysts and blastocyst outgrowths. After penetration of the zona, the fertilizing spermatozoon showed intense fluorescence upon fusion with the vitelline membrane. Due to fluorochrome leakage from the perivitelline space a faint fluorescence was detected in zona-bound spermatozoa. This fluorescence of zona-bound spermatozoa intensified with increased fluorochrome concentration (10 micrograms/ml), obscuring the fluorescence of the fertilizing spermatozoa. Spermatozoa added to zona-free mouse oocytes (pre-loaded with 1 or 10 micrograms H-33342/ml) fluoresced within 10 min of insemination, provided the zonae were removed mechanically. Removal by protease digestion induced leakage of fluorochrome, so that all spermatozoa in the vicinity of an oocyte pre-loaded with 10 micrograms H-33342/ml became labelled. This leakage was not visibly apparent when protease-treated oocytes were exposed to only 1 microgram H-33342/ml. The technique could not be applied to zona-free hamster oocytes under our conditions, since the fluorochrome leaked freely from the oocytes whether the zona was removed mechanically or enzymically.