Divergent functions of the proneural genes Mash1 and Ngn2 in the specification of neuronal subtype identity.

The neural bHLH genes Mash1 and Ngn2 are expressed in complementary populations of neural progenitors in the central and peripheral nervous systems. Here, we have systematically compared the activities of the two genes during neural development by generating replacement mutations in mice in which the coding sequences of Mash1 and Ngn2 were swapped. Using this approach, we demonstrate that Mash1 has the capacity to respecify the identity of neuronal populations normally derived from Ngn2-expressing progenitors in the dorsal telencephalon and ventral spinal cord. In contrast, misexpression of Ngn2 in Mash1-expressing progenitors does not result in any overt change in neuronal phenotype. Taken together, these results demonstrate that Mash1 and Ngn2 have divergent functions in specification of neuronal subtype identity, with Mash1 having the characteristics of an instructive determinant whereas Ngn2 functions as a permissive factor that must act in combination with other factors to specify neuronal phenotypes. Moreover, the ectopic expression of Ngn2 can rescue the neurogenesis defects of Mash1 null mutants in the ventral telencephalon and sympathetic ganglia but not in the ventral spinal cord and the locus coeruleus, indicating that Mash1 contribution to the specification of neuronal fates varies greatly in different lineages, presumably depending on the presence of other determinants of neuronal identity.

A novel use of alginate hydrogel as Schwann cell matrix.

The use of bioresorbable conduits supplemented with Schwann cells (SCs) is a promising tissue engineering technique to replace nerve grafting. Alginate hydrogel (AH), as a SC tissue engineering matrix, has many advantages over previously used matrices but has not been evaluated for this purpose. In this study, the viability and proliferation of SCs together with SC function in AH was evaluated in vitro. AlamarBlue cell assay was used to monitor the viability of SCs in AH and compared to SC viability in collagen gel, fibrin glue, hyaluronic acid, Matrigel, and standard culture plate over 5 days in culture. The results showed that the viability and growth of SCs in different matrices over the culture period did not significantly differ to culture plate culture. SC function when suspended in AH was monitored using chick embryo dorsal root ganglia (CDRG) growth assay. Growth of CDRG in AH with or without SCs was compared to CDRG growth without AH matrix. After 3 days in culture, the mean length of neurite sprouting was measured. The results showed that there was neurite growth in AH but was reduced to 43% of control. The neurite growth in AH was, however, enhanced by 170% when SCs were suspended in the gel. In conclusion, AH supported SC viability and function in vitro and may be useful in peripheral nerve tissue engineering in reconstructive procedures.

Directed and enhanced neurite growth with pulsed magnetic field stimulation.

Pulsed magnetic field (PMF) stimulation was applied to mammalian neurons in vitro to influence axonal growth and to determine whether induced current would direct and enhance neurite growth in the direction of the current. Two coils were constructed from individual sheets of copper folded into a square coil. Each coil was placed in a separate water-jacketed incubator. One was energized by a waveform generator driving a power amplifier, the other was not energized. Whole dorsal root ganglia (DRG) explant cultures from 15-day Sprague-Dawley rat embryos were established in supplemented media plus nerve growth factor (NGF) at concentrations of 0-100 ng/mL on a collagen-laminin substrate. Dishes were placed at the center of the top and bottom of both coils, so that the DRG were adjacent to the current flowing in the coil. After an initial 12 h allowing DRG attachment to the substrate floor, one coil was energized for 18 h, followed by a postexposure period of 18 h. Total incubation time was 48 h for all DRG cultures. At termination, DRG were histochemically stained for visualization and quantitative analysis of neurite outgrowth. Direction and length of neurite outgrowth were recorded with respect to direction of the current. PMF exposed DRG exhibited asymmetrical growth parallel to the current direction with concomitant enhancement of neurite length. DRG cultures not PMF exposed had a characteristic radial pattern of neurite outgrowth. These results suggest that PMF may offer a noninvasive mechanism to direct and promote nerve regeneration.

Expression domains of murine ephrin-A5 in the pituitary and hypothalamus.

Using in situ hybridization on whole-mounts and sections of mouse embryos we have visualized the pattern of expression for the Eph receptor ligand ephrin-A5. Non neuronal expression domains include the ectoderm of the branchial arches, the ectoderm and mesenchyme surrounding the dorsal root ganglia, the intervertebral discs, maxillary and mandibulary mesenchymal elements as well as the nasal mesenchyme and ectoderm. Within the developing nervous system, ephrin-A5 expression is very dynamic. Besides the midbrain it is also expressed in the hypothalamus, and the neurohypophysis that we studied here in more detail. Hypothalamus expression of ephrin-A5 demarks distinct nuclei, persists throughout embryonic development, and can be seen also in the adult.

[The neurite-outgrowth promoting effect from Clarke's nucleus of cat spinal cord with spared dorsal root preparation under electro-acupuncture stimulation on dorsal root ganglion of chicken embryo].

A modification of the double-coverslip hanging-drop culture technique originated from Maximow was introduced in the present report. The neurite - outgrowth promoting effect from Clarke's nucleus of cat spinal cord with partially lumbosacral dorsal rhizotomy as well as acupuncture stimulation of the points located within the peripheral innervating field of the spared dorsal root was examined with the modified culture technique. Findings demonstrated that an increased neurite-outgrowth promoting effect appeared in Clarke's nucleus of cat spinal cord with partial deafferentation, and that electro-acupuncture stimulation could enhance the neurite-outgrowth promoting effect further. It suggested that the increment of the neurite-outgrowth promoting effect might be related to the collateral sprouting of spared dorsal root and to the sprouting-promotion effect of acupuncture stimulation.

Jagged: a mammalian ligand that activates Notch1.

Here we report the isolation of a rat cDNA clone, Jagged, which we show encodes a ligand for vertebrate Notch. Our conclusion is based on three observations. First, sequence analysis reveals substantial homology between Jagged and invertebrate ligands for the LIN-12/Notch proteins. Second, in situ hybridization of rat embryos identifies both distinct and overlapping patterns of gene expression for Jagged with those for Notch1, Notch2, and Notch3. Finally, the biological activity of Jagged was tested using a cell culture assay in which Jagged activates rat Notch1 expressed in myoblasts and prevents muscle cell differentiation. Our data support the hypothesis that Notch-ligand interactions function in maintaining mammalian cells in an undifferentiated state.

The development of cutaneous afferent pathways in fetal sheep: a structural and functional study.

In this study we have examined the functional and structural development of cutaneous afferent pathways in the fetal sheep hindlimb from 67 to 143 days of gestation. The earliest age at which extracellular discharges could be evoked in dorsal root ganglia and in dorsal horn cells by natural cutaneous stimulation was 75 days. The majority of cells responded to light stroking or indentation of the skin (low threshold) although some cells responded to intense squeezing (high threshold). With increasing gestational age, the majority of cells continued to respond to low threshold stimuli with cells responding to intense mechanical stimuli being recorded less frequently. Dorsal root ganglion cells responding to the cutaneous application of noxious heat (> 45 degrees C) were first observed at 107 days. We have also shown that fibres projecting from dorsal horn cells and presumably entering the spinothalamic tract carry signals at least as far as the mid thoracic spinal cord by 104 days. Ultrastructural studies of the skin revealed bundles of unmyelinated axons located to blood vessels throughout the dermis at 68 days. Innervation of the skin was first observed by silver staining at 75 days when fibres could be seen running along blood vessels in the dermis. With increasing gestational age fibres were seen traversing the dermis to innervate the epidermis as free nerve endings. Wool/hair follicle innervation was first evident at about 100-106 days with only a few follicles being innervated at this age. By 115 days, nerve endings had begun to form circumferential wrappings around follicles and by 134 days lanceolate terminals were observed around the base of the follicles. Biocytin-labelled afferent fibres entered the dorsal horn at 56 days. This initial innervation was sparse but by 76 days there was a marked increase in both the number of afferent fibres entering the dorsal horn and in the extent of their arborisation. Ultrastructural studies revealed that terminals immunoreactive for calcitonin gene-related peptide were present in lamina I as early as 61 days. The period of maximal synaptogenesis and synaptic maturation of this group of terminals appeared to occur between 87-128 days. Therefore the commencement of activation of dorsal horn cells by natural cutaneous stimulation occurs by mid gestation (75 days) in the fetal sheep. This is at the same time or just after peripheral nerves first innervate the skin and about 2 weeks after primary afferent terminals can be identified in the dorsal horn.

Naturally occurring and induced neuronal death in the chick embryo in vivo requires protein and RNA synthesis: evidence for the role of cell death genes.

Treatment of chick embryos in ovo for 10-12 hr with inhibitors of protein and RNA synthesis during the peak time of normal cell death (Embryonic Day 8) for motoneurons and dorsal root ganglion cells markedly reduces the number of degenerating neurons in these populations. The massive neuronal death induced by the early absence of the limbs was also blocked almost completely by these agents. Further, the death of neurons following peripheral axotomy at the end of the normal cell death period (Embryonic Day 10) was reduced significantly by treatment with inhibitors of biosynthetic reactions. These results indicate that, in vivo, naturally occurring neuronal death, neuronal death induced by the absence of peripheral targets, and axotomy-induced neuronal death later in development all require active gene expression and protein and RNA synthesis. Therefore, neuronal death in a variety of situations may reflect the expression of a developmental fate that can normally only be overridden or suppressed by specific environmental signals (e.g., neurotrophic molecules).

Toxic effects of organophosphates on nerve cell growth and ultrastructure in culture.

Organophosphates (OP) comprise one of the major classes of pesticides in use today. It is well accepted that the primary site of action of the OPs is at cholinergic synapses. However, it has been suggested that OPs may have direct neural effects as well. In this study, cultured chick dorsal root ganglia (DRG) were used to study the effect of fenthion (FEN), an OP pesticide, on isolated nerve cell growth and ultrastructure. Light microscopic evaluation revealed a dose-response relationship between the concentration of FEN (10(-2) M to 10(-5) M) and severity of morphologic changes. Cultured explants were treated with a lower concentration of FEN (10(-6) M) and morphologic alterations were compared to those observed in explants treated with 10(-6) M paraoxon, a more acutely toxic OP, or 10(-6) M neostigmine, a non-OP inhibitor of acetyl-cholinesterase. Based on both light and electron microscopy, neostigmine had no observed effect on cell morphology except for an inhibition of the extension of neurites by DRG cells. In contrast, explants treated with OPs exhibited a significant alteration in cell morphology. Initial lesions were observed first in the neurites and pseudopodia and consisted of vacuolization, loss of tubular structures, retraction of pseudopodia, and cell membrane disruption at the growth cone. Lipid accumulations were observed within the cytoplasm of treated cells. The effects of paraoxon on DRG cell morphology were significantly more severe than the effects of FEN, and lipid vacuoles observed in paraoxon-treated cells were several times larger than those observed in FEN-treated cells (5-10 microns in diameter vs. 0.5-1.0 microns in diameter). Results show that OPs have a direct effect on DRG nerve cells in culture, consistent with an alteration in cell membrane integrity. Cultured DRG cells can be useful in the evaluation of toxicologic effects.

Differentiation of postmitotic neuroblasts into substance P-immunoreactive sensory neurons in dissociated cultures of chick dorsal root ganglion.

Counts performed on dissociated cell cultures of E10 chick embryo dorsal root ganglia (DRG) showed after 4-6 days of culture a pronounced decline of the neuronal population in neuron-enriched cultures and a net gain in the number of ganglion cells in mixed DRG cell cultures (containing both neurons and nonneuronal cells). In the latter case, the increase in the number of neurons was found to depend on NGF and to average 119% in defined medium or 129% in horse serum-supplemented medium after 6 days of culture. The lack of [3H]thymidine incorporation into the neuronal population indicated that the newly formed ganglion cells were not generated by proliferation. On the contrary, the differentiation of postmitotic neuroblasts present in the nonneuronal cell compartment was supported by sequential microphotographs of selected fields taken every hour for 48-55 hr after 3 days of culture. Apparently nonneuronal flat dark cells exhibited morphological changes and gradually evolved into neuronal ovoid and refringent cell bodies with expanding neurites. The ultrastructural organization of these evolving cells corresponded to that of primitive or intermediate neuroblasts. The neuronal nature of these rounding up cell bodies was indeed confirmed by the progressive expression of various neuronal cell markers (150 and 200-kDa neurofilament triplets, neuron specific enolase, and D2/N-CAM). Besides a constant lack of immunoreactivity for tyrosine hydroxylase, somatostatin, parvalbumin, and calbindin-D 28K and a lack of cytoenzymatic activity for carbonic anhydrase, all the newly produced neurons expressed three main phenotypic characteristics: a small cell body, a strong immunoreactivity to MAG, and substance P. Hence, ganglion cells newly differentiated in culture would meet characteristics ascribed to small B sensory neurons and more specifically to a subpopulation of ganglion cells containing substance P-immunoreactive material.

Schwann cell myelination in a chemically defined medium: demonstration of a requirement for additives that promote Schwann cell extracellular matrix formation.

Primary cultures of embryonic rat Schwann cells and sensory nerve cells can be grown in the serum-free defined medium N2, but the Schwann cells fail to deposit extracellular matrix and do not ensheath or myelinate axons. Previously these functions could be induced only in media supplemented with serum and chick embryo extract. Here we show that supplementing N2 medium with ascorbic acid and a commercial preparation of the glycoprotein fetuin or purified bovine serum albumin in the absence of serum or other undefined media components leads to increased production of Schwann cell extracellular matrix and extensive myelin formation by Schwann cells. Ascorbic acid is required for production of collagen type IV. Both ascorbic acid and one of the proteins are required for optimal extracellular matrix formation and myelination. These results lend support to the hypothesis that production of extracellular matrix by Schwann cells is necessary for myelination of nerve fibers.