NGF-TrkA Signaling by Sensory Nerves Coordinates the Vascularization and Ossification of Developing Endochondral Bone.

Developing tissues dictate the amount and type of innervation they require by secreting neurotrophins, which promote neuronal survival by activating distinct tyrosine kinase receptors. Here, we show that nerve growth factor (NGF) signaling through neurotrophic tyrosine kinase receptor type 1 (TrkA) directs innervation of the developing mouse femur to promote vascularization and osteoprogenitor lineage progression. At the start of primary ossification, TrkA-positive axons were observed at perichondrial bone surfaces, coincident with NGF expression in cells adjacent to centers of incipient ossification. Inactivation of TrkA signaling during embryogenesis in TrkA(F592A) mice impaired innervation, delayed vascular invasion of the primary and secondary ossification centers, decreased numbers of Osx-expressing osteoprogenitors, and decreased femoral length and volume. These same phenotypic abnormalities were observed in mice following tamoxifen-induced disruption of NGF in Col2-expressing perichondrial osteochondral progenitors. We conclude that NGF serves as a skeletal neurotrophin to promote sensory innervation of developing long bones, a process critical for normal primary and secondary ossification.

Specification of dopaminergic subsets involves interplay of En1 and Pitx3.

Mesodiencephalic dopaminergic (mdDA) neurons control locomotion and emotion and are affected in multiple psychiatric and neurodegenerative diseases, including Parkinson's disease (PD). The homeodomain transcription factor Pitx3 is pivotal in mdDA neuron development and loss of Pitx3 results in programming deficits in a rostrolateral subpopulation of mdDA neurons destined to form the substantia nigra pars compacta (SNc), reminiscent of the specific cell loss observed in PD. We show here that in adult mice in which the gene encoding a second homeoprotein, engrailed 1 (En1), has been deleted, dramatic loss of mdDA neurons and striatal innervation defects were observed, partially reminiscent of defects observed in Pitx3(-/-) mice. We then continue to reveal developmental crosstalk between En1 and Pitx3 through genome-wide expression analysis. During development, both En1 and Pitx3 are required to induce expression of mdDA genes in the rostrolateral subset destined to form the SNc. By contrast, Pitx3 and En1 reciprocally regulate a separate gene cluster, which includes Cck, demarcating a caudal mdDA subset in wild-type embryos. Whereas En1 is crucial for induction of this caudal phenotype, Pitx3 antagonizes it rostrolaterally. The combinatorial action of En1 and Pitx3 is potentially realized through at least three levels of molecular interaction: (1) influencing each other's expression level, (2) releasing histone deacetylase-mediated repression of Nurr1 target genes and (3) modulating En1 activity through Pitx3-driven activation of En1 modulatory proteins. These findings show how two crucial mediators of mdDA neuronal development, En1 and Pitx3, interact in dopaminergic subset specification, the importance of which is exemplified by the specific vulnerability of the SNc found in PD.

An autocrine Wnt5a-Ror signaling loop mediates sympathetic target innervation.

During nervous system development, axon branching at nerve terminals is an essential step in the formation of functional connections between neurons and target cells. It is known that target tissues exert control of terminal arborization through secretion of trophic factors. However, whether the in-growing axons themselves produce diffusible cues to instruct target innervation remains unclear. Here, we use conditional mutant mice to show that Wnt5a derived from sympathetic neurons is required for their target innervation in vivo. Conditional deletion of Wnt5a resulted in specific deficits in the extension and arborization of sympathetic fibers in their final target fields, while no defects were observed in the overall tissue patterning, proliferation, migration or differentiation of neuronal progenitors. Using compartmentalized neuronal cultures, we further demonstrate that the Ror receptor tyrosine kinases are required locally in sympathetic axons to mediate Wnt5a-dependent branching. Thus, our study suggests an autocrine Wnt5a-Ror signaling pathway that directs sympathetic axon branching during target innervation.

ATX expression and LPA signalling are vital for the development of the nervous system.

Autotaxin (ATX) is a secreted glycoprotein widely present in biological fluids, originally isolated from the supernatant of melanoma cells as an autocrine motility stimulation factor. Its enzymatic product, lysophosphatidic acid (LPA), is a phospholipid mediator that evokes growth-factor-like responses in almost all cell types through G-protein coupled receptors. To assess the role of ATX and LPA signalling in pathophysiology, a conditional knockout mouse was created. Ubiquitous, obligatory deletion resulted to embryonic lethality most likely due to aberrant vascular branching morphogenesis and chorio-allantoic fusion. Moreover, the observed phenotype was shown to be entirely depended on embryonic, but not extraembryonic or maternal ATX expression. In addition, E9.5 ATX null mutants exhibited a failure of neural tube closure, most likely independent of the circulatory failure, which correlated with decreased cell proliferation and increased cell death. More importantly, neurite outgrowth in embryo explants was severely compromised in mutant embryos but could be rescued upon the addition of LPA, thus confirming a role for ATX and LPA signalling in the development of the nervous system. Finally, expression profiling of mutant embryos revealed attenuated embryonic expression of HIF-1a in the absence of ATX, suggesting a novel effector pathway of ATX/LPA.

Human fetal neural stem cells in rat brain: effects of preculturing and transplantation.

The fate of human fetal stem/progenitor cells transplanted into rat brain depends on conditions of preculturing (long or short) and state and site of transplantation. Human nestin-positive stem cells cultured according to the short protocol did not migrate into hypoxic and normal brain after transplantation, but actively migrated in damaged spinal cord. After transplantation of long-cultured cells into the brain mainly committed neuroblasts and solitary nestin-positive cells migrated from the site of transplantation into the brain.

Generation of purified oligodendrocyte progenitors from embryonic stem cells.

Demyelination is a key component in the pathogenesis of many neurological disorders. Transplantation of myelinating cells may offer a therapeutic approach to restore neurological function in these diseases. Recent findings suggest that pluripotent embryonic stem (ES) cells can serve as an unlimited donor source for neural transplantation. The clinical application of ES cells for myelin repair will depend critically on the ability to enrich oligodendroglial progenitors in high purity. Combining controlled differentiation in the presence of growth factors and genetic lineage selection, we devised a cell culture protocol yielding highly purified oligodendrocyte progenitors. Murine ES cell clones stably transfected with a construct encoding the beta-galactosidase-neomycine phosphotransferase fusion protein (beta(geo)) under control of the 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNP) promoter were differentiated into bipotential glial precursors. Subsequent induction of a CNP-positive stage and selection in neomycine resulted in a homogenous cell population with a pre-oligodendrocyte phenotype. The selected cells continued to proliferate in the presence of FGF-2 and PDGF and, upon growth factor withdrawal, differentiated into mature galactocerebroside (GalC)-positive oligodendrocytes. Transplantation studies in myelin-deficient (md) rats indicate that ES cell-derived oligodendrocyte progenitors generated with this method may serve as an attractive donor source for myelin repair.

Live view of gonadotropin-releasing hormone containing neuron migration.

Neurons that synthesize GnRH control the reproductive axis and migrate over long distances and through different environments during development. Prior studies provided strong clues for the types of molecules encountered and movements expected along the migratory route. However, our studies provide the first real-time views of the behavior of GnRH neurons in the context of an in vitro preparation that maintains conditions comparable to those in vivo. The live views provide direct evidence of the changing behavior of GnRH neurons in their different environments, showing that GnRH neurons move with greater frequency and with more changes in direction after they enter the brain. Perturbations of guiding fibers distal to moving GnRH neurons in the nasal compartment influenced movement without detectable changes in the fibers in the immediate vicinity of moving GnRH neurons. This suggests that the use of fibers by GnRH neurons for guidance may entail selective signaling in addition to mechanical guidance. These studies establish a model to evaluate the influences of specific molecules that are important for their migration.

Targeting of neuromodulin (GAP-43) fusion proteins to growth cones in cultured rat embryonic neurons.

Neuromodulin (GAP-43) is a membrane protein that is transported to neuronal growth cones. Zuber and co-workers have proposed that the N-terminal 10 amino acid sequence of neuromodulin is sufficient to target proteins to growth cones. We demonstrate that a neuromodulin-beta-galactosidase fusion protein is transported to growth cones of cultured rat neurons, whereas a fusion protein containing the N-terminal 10 amino acids of neuromodulin and beta-galactosidase is not. A mutant neuromodulin lacking cysteines 3 and 4, the palmitylation sites required for membrane attachment, does not target beta-galactosidase to growth cones. We conclude that membrane attachment is required for growth cone accumulation and that structural elements, in addition to the first 10 amino acids of neuromodulin, may be required for growth cone targeting.

Initial development of capillaries in the neuroepithelium of the mouse.

The developmental stage of the mouse embryo at which capillaries first occurred and their localisation in the neuroepithelium were investigated on serial sections of 9 and 10 days old embryos embedded in paraffin and Epon. In addition, areas of the neuroepithelium in which capillaries had been observed at the light microscopical level were investigated by electron microscopy carried out on embryos fixed with glutaraldehyde supplemented with tannic acid. In 5-7 microns serial sections of paraffin-embedded embryos, capillaries were initially seen in the CNS anlage at Theiler's Stage 14 (1972). At this stage, capillaries also occurred in the prosencephalon, the rhombencephalon and in the developing spinal cord. In 1 micron serial sections of resin-embedded embryos, capillaries could be identified in the neuroepithelium one stage earlier, i.e. at Stage 13. These very early capillaries were seen in the dorsal part of the lateral wall of the prosencephalon which later forms the diencephalon. At the ultrastructural level, those areas of the neuroepithelium in which leptomeningeal capillaries first started to spread into the neuroepithelium were characterised by the disintegration of the basement membrane of the neuroepithelium and the appearance of tannic acid-positive extracellular structures between the neuroepithelium and the adjacent leptomeningeal capillaries.

Ontogeny of this immuno-neuro-endocrine relationship. Changes in lymphoid tissue of chick embryos surgically decapitated at 33-38 hours of incubation.

The prosencephalon, and primordium of the hypophysis were surgically removed from chick embryos at 33-38 hours of incubation. The thymus, bursa of Fabricius, spleen, bone marrown and liver were examined cytomorphologically on day 15, 17 and 19. T marker-bearing and Bu marker-bearing lymphocytes were identified by immunofluorescence. Decapitated embryos tended to be smaller than sham-decapitated controls of the same age, and exhibited retarded development of the thymus, bursa, spleen and liver. Decapitation particularly affected the cellular composition of the bursa and spleen, induced a decrease in the number of lymphocytes, and caused a striking depletion of lymphocytes bearing Bu antigen. This experiment showed an interdependence between lymphoid (immune), nervous and endocrine centers in the chick embryo.