Neuronal competition determines the spatial pattern of neuropeptide expression by identified neurons of the leech.

Staining adult and embryonic leech ventral nerve cords with antibodies raised against the molluscan neuropeptides small cardioactive peptide B (SCP) and FMRFamide results in segment-specific and bilaterally asymmetric patterns of cell staining. One immunoreactive neuron, the RAS interneuron, is present in only four rostral segmental ganglia, while another, the CAS interneuron, is restricted to the four most caudal abdominal ganglia and tail. In addition to their segment-specific distributions, only one RAS or CAS cell is found in each segmental ganglion, and they alternate sides between adjacent ganglia (either L-R-L-R or R-L-R-L) with a fidelity of about 95%. This paper utilizes cell deletion techniques to investigate the determination of the asymmetric and alternating pattern of RAS and CAS neurons. We show that developmentally equivalent RAS and CAS homologs are present on both sides of the appropriate ganglia, and that within each ganglion one of the initially paired homologs loses the ability to assume the immunoreactive RAS or CAS fate 2-3 days after axonogenesis has begun. These experiments suggest that there is a competitive interaction between bilateral homologs which ensures that only one mature RAS/CAS neuron is formed per ganglion, and that contralateral RAS/CAS neurons are not required in the same or adjacent ganglia for the determination of the RAS or CAS developmental pathways. Nerve cord transections between ganglia in the CAS domain can alter the spatial pattern of CAS neuron determination, confirming that both bilateral homologs retain the ability to express neuropeptide until late embryonic stages, and suggesting that the alternating pattern of RAS/CAS cells requires communication between adjacent ganglia through the longitudinal connectives.     

Auditory hair cell explant co-cultures promote the differentiation of stem cells into bipolar neurons

Auditory neurons, the target neurons of the cochlear implant, degenerate following a sensorineural hearing loss. The goal of this research is to direct the differentiation of embryonic stem cells (SCs) into bipolar auditory neurons that can be used to replace degenerating neurons in the deafened mammalian cochlea. Successful replacement of auditory neurons is likely to result in improved clinical outcomes for cochlear implant recipients. We examined two post-natal auditory co-culture models with and without neurotrophic support, for their potential to direct the differentiation of mouse embryonic SCs into characteristic, bipolar, auditory neurons. The differentiation of SCs into neuron-like cells was facilitated by co-culture with auditory neurons or hair cell explants, isolated from post-natal day five rats. The most successful combination was the co-culture of hair cell explants with whole embryoid bodies, which resulted in significantly greater numbers of neurofilament-positive, neuron-like cells. While further characterization of these differentiated cells will be essential before transplantation studies commence, these data illustrate the effectiveness of post-natal hair cell explant co-culture, at providing valuable molecular cues for directed differentiation of SCs towards an auditory neuron lineage.     

Calbindin immunoreactivity of enteric neurons in the guinea-pig ileum

Previous studies have identified Dogiel type II neurons with cell bodies in the myenteric plexus of guinea-pig ileum to be intrinsic primary afferent neurons. These neurons also have distinctive electrophysiological characteristics (they are AH neurons) and 82-84% are immunoreactive for calbindin. They are the only calbindin-immunoreactive neurons in the plexus. Neurons with analogous shape and electrophysiology are found in submucosal ganglia, but, with antibodies used in previous studies, they lack calbindin immunoreactivity. An antiserum that is more effective in revealing calbindin in the guinea-pig enteric nervous system has been reported recently. In the present work, we found that this antiserum reveals the same population that was previously identified in myenteric ganglia, and does not reveal any further population of myenteric nerve cells. In submucosal ganglia, 9-10% of nerve cells were calbindin immunoreactive with this antiserum. The submucosal neurons with calbindin immunoreactivity were also immunoreactive for choline acetyltransferase, but not for neuropeptide Y (NPY) or vasoactive intestinal peptide (VIP). Small calbindin-immunoreactive neurons (average profile 130 microm2) were calretinin immunoreactive, whereas the large calbindin-immunoreactive neurons (average profile 330 microm2) had tachykinin (substance P) immunoreactivity. Calbindin immunoreactivity was seen in about 50% of the calretinin neurons and 40% of the tachykinin-immunoreactive submucosal neurons. It is concluded that, in the guinea-pig ileum, only one class of myenteric neuron, the AH/Dogiel type II neuron, is calbindin immunoreactive, but, in the submucosal ganglia, calbindin immunoreactivity occurs in cholinergic, calretinin-immunoreactive, secretomotor/vasodilator neurons and AH/Dogiel type II neurons.     

Identification of the populations of enteric neurons that have NK1 tachykinin receptors in the guinea-pig small intestine

Simultaneous immunofluorescence labelling was used to investigate the patterns of colocalisation of the NK1 tachykinin receptor with other neuronal markers, and hence determine the functional classes of neuron that bear the NK1 receptor in the guinea-pig ileum. In the myenteric plexus, 85% of NK1 receptor-immunoreactive (NK1r-IR) nerve cells had nitric oxide synthase (NOS) immunoreactivity and the remaining 15% were immunoreactive for choline acetyltransferase (ChAT). Of the latter group, about 50% were immunoreactive for both neuropeptide Y (NPY) and somatostatin (SOM), and had the morphologies of secretomotor neurons. Many of the remaining ChAT neurons were immunoreactive for calbindin or tachykinins (TK), but not both. These calbindin immunoreactive neurons had Dogiel type II morphology. No NK1r-IR nerve cells in the myenteric plexus had serotonin or calretinin immunoreactivity. In the submucosal ganglia, 84% of NK1r-IR nerve cells had neuropeptide Y immunoreactivity and 16% were immunoreactive for TK. It is concluded that NK1r-IR occurs in five classes of neuron; namely, in the majority of NOS-immunoreactive inhibitory motor neurons, in ChAT/TK-immunoreactive excitatory neurons to the circular muscle, in all ChAT/NPY/SOM-immunoreactive secretomotor neurons, in a small proportion of ChAT/calbindin myenteric neurons, and in about 50% of ChAT/TK submucosal neurons.     

S100 is present in developing chicken neurons and Schwann cells and promotes motor neuron survival in vivo.

We used polyclonal antisera recognizing S100, a small acidic protein highly enriched in nervous tissue, to stain sections of embryonic chicken lumbosacral spinal cord and hindlimb. S100 immunoreactivity was detected in developing sensory neurons of the dorsal root ganglia (DRG) and motor neurons of the ventral spinal cord as early as embryonic day (E) 5, and staining persisted through hatching. In contrast, expression of S100 first became apparent in Schwann cells at E13, just before myelination, and was not detected in developing skin or muscle. Since S100 beta was present in motor and sensory neurons and is known to promote neuronal survival and neurite extension in vitro (Winningham-Major, Staecker, Barger, Coats, and Van Eldik, 1989), we tested the ability of S100 to promote neuron survival in an in ovo survival assay. Addition of S100 to chick embryos in ovo during the period of naturally occurring motor neuron cell death resulted in a significant increase in motor neuron survival, but had no effect on the in vivo survival of sensory neurons in the DRG. The findings that S100 is present in spinal motor neurons and that the addition of S100 enhances the survival of these cells in vivo are consistent with the possibility that S100 may act as a naturally occurring neuron survival factor during development.     

Ontogenic attendance of neuropeptides in the embryo chicken retina

We have examined the ontogeny of somatostatin-, Glucagon-, Vasoactive Intestinal Polypeptide-, Substance P-, Neuropeptide Y, and Calcitonin gene-related peptide-Iike structures in the chicken retina by immunocytochemistry. Neuroblastic cells containing Substance P-Iike immunoreactivity (IR) first appeared at embryonic day 5 in the peripheral portion of the retina. Somatostatin-like immunoreactivity was detected as early as embryonic day 11 in the innermost level of the inner neuroblastic layer. The distribution pattern of amacrine cells containing Vasoactive Intestinal Peptide-Iike immunoreactivity was similar to that for Neuropeptide Y- and Calcitonin gene-related peptide-Iike immunoreactive cells. These three types of IR cell appeared at embryonic day 13. Glucagon-like immunoreactive cells first appeared in the retina at embryonic day 15, in the innermost part of the inner nuclear layer. From the 13th to 15th day of incubation, the number and intensity of Calcitonin gene-related peptide-, Somatostatin-, Neuropeptide Y- and Substance P-Iike immunoreactive cells increased and then decreased progressively before hatching. Glucagon immunoreactive cells increased in number on the last day before hatching. After embryonic day 15, the amacrine cells containing Vasoactive intestinal peptide-Iike immunoreactivity decreased notably in number. Our study showed that development of these immunoreactive structures was different for each neuropeptide. These differences in development may reflect the diverse neurophysiological roles of these neuroactive peptides, which could act as neurotransmitters/neuromodulators at the chick retinal level. Their presence may indicate roles as neuronal differentiation or growth factors.     

Immunocytochemical study on photoreceptor cell differentiation in the cultured retina of the chick

Photoreceptor cell differentiation was investigated in a dissociated monolayer culture of chick embryonic retinas with electron microscopic immunohistochemistry. The antibody was raised against bovine rhodopsin purified on SDS-polyacrylamide gel electrophoresis. In the developing retina, immunoreactivity was first recognized on the 14th day of incubation and was localized on the plasma membrane of the growing inner segment. On the 16th day, immunoreactivity was observed on some differentiating outer segments but not on inner segments. In the culture from 6 1/2-day-old embryonic retinas, immunoreactivity was found on the 7th day of culturing on the plasma membrane of large-sized neurons. Electron microscopic observations confirmed that such stained cells showed reaction product on the plasma membrane, and that they displayed fine structures characteristic of intact photoreceptor cells. They had a number of microvillous processes and often one thick process, both of which were intensely stained. Outer segment formation, however, was not observed in the present monolayer culture. These results indicate that opsin synthesis and its transport to the plasma membrane begins prior to and probably independently of outer segment formation.     

Distribution of substance P immunoreactive cell bodies and fibers in cranial sensory and autonomic ganglia of the chick

Substance P-immunoreactive neurons were demonstrated in chick embryonic and adult trigeminal ganglion and jugular-superior ganglionic complex using FITC-immunohistochemical methods. Both small-size and large ganglion cells exhibited SP immunoreactivity, without apparent changes during embryonic and post-hatching development. SP-positive fibers could be detected in a good number in the sympathetic cranial cervical ganglion, either during embryonic development or in adult chick. No immunoreactive perikarya were observed in this ganglion. In the ciliary ganglion, both choroidal and ciliary neurons were SP-negative, whereas SP immunoreactive fibers surrounded the perikarya of both cell populations.     

S100 is present in developing chicken neurons and schwann cell and promotes motor neuron survival in vivo

We used polyclonal antisera recognizing S100, a small acidic protein highly enriched in nervous tissue, to stain sections of embryonic chicken lumbosacral spinal cord and hindlimb. S100 immunoreactivity was detected in developing sensory neurons of the dorsal root ganglia (DRG) and motor neurons of the ventral spinal cord as early as embryonic day (E) 5, and staining persisted through hatching. In contrast, expression of S100 first became apparent in Schwann cells at E13, just before myelination, and was not detected in developing skin or muscle. Since S100β was present in motor and sensory neurons and is known to promote neuronal survival and neurite extension in vitro (Winningham-Major, Staecker, Barger, Coats, and Van Eldik, 1989), we tested the ability of S100 to promote neuron survival in an in ovo survival assay. Addition of S100 to chick embryos in ovo during the period of naturally occurring motor neuron cell death resulted in a significant increase in motor neuron survival, but had no effect on the in vivo survival of sensory neurons in the DRG. The findings that S100 is present in spinal motor neurons and that the addition of S100 enhances the survival of these cells in vivo are consistent with the possibility that S100 may act as a naturally occurring neuron survival factor during development. © 1992 John Wiley & Sons, Inc.     

A Genome-Wide RNAi Screen for Factors Involved in Neuronal Specification in Caenorhabditis elegans

One of the central goals of developmental neurobiology is to describe and understand the multi-tiered molecular events that control the progression of a fertilized egg to a terminally differentiated neuron. In the nematode Caenorhabditis elegans, the progression from egg to terminally differentiated neuron has been visually traced by lineage analysis. For example, the two gustatory neurons ASEL and ASER, a bilaterally symmetric neuron pair that is functionally lateralized, are generated from a fertilized egg through an invariant sequence of 11 cellular cleavages that occur stereotypically along specific cleavage planes. Molecular events that occur along this developmental pathway are only superficially understood. We take here an unbiased, genome-wide approach to identify genes that may act at any stage to ensure the correct differentiation of ASEL. Screening a genome-wide RNAi library that knocks-down 18,179 genes (94% of the genome), we identified 245 genes that affect the development of the ASEL neuron, such that the neuron is either not generated, its fate is converted to that of another cell, or cells from other lineage branches now adopt ASEL fate. We analyze in detail two factors that we identify from this screen: (1) the proneural gene hlh-14, which we find to be bilaterally expressed in the ASEL/R lineages despite their asymmetric lineage origins and which we find is required to generate neurons from several lineage branches including the ASE neurons, and (2) the COMPASS histone methyltransferase complex, which we find to be a critical embryonic inducer of ASEL/R asymmetry, acting upstream of the previously identified miRNA lsy-6. Our study represents the first comprehensive, genome-wide analysis of a single neuronal cell fate decision. The results of this analysis provide a starting point for future studies that will eventually lead to a more complete understanding of how individual neuronal cell types are generated from a single-cell embryo.     

Immunocytochemical study using a GABA antiserum for the demonstration of inhibitory neurons in the rat locus ceruleus

The localization of GABA-like immunoreactivity in the locus ceruleus of rats was studied by the peroxidase-antiperoxidase (PAP) method using a purified antibody raised against GABA applied to paraffin sections, with counterstaining by cresylecht violet, and to floating sections for preembedding immunoelectron microscopy. A few medium-sized and some small neurons showed GABA-like immunoreactivity in both nuclei and perikarya. The preferential localization of these immunopositive neurons in the marginal parts of the locus ceruleus suggests that they are inhibitory local circuit neurons located between this center and the afferent fiber systems. Some of the immunoreactive neurons displayed homogeneous and heterogeneous "paired cells" patterns. Occurrence of the GABA-GABA interaction is indicated. Immunopositive bouton forms are located close to every positive and negative neuron. Electron microscopy confirms GABA-like immunoreactivity in both medium-sized and small neurons of the locus ceruleus and demonstrates that immunoreactive boutons are axosomatic and axosoma spine symmetric synapses on immunopositive and immunonegative cell bodies. These immunocytochemical results support the existence of inhibitory interneurons in the locus ceruleus.     

Coexpression of sensory and autonomic neurotransmitter traits by avian neural crest cells in vitro.

This study shows that explants of quail neural crest cultured in a medium containing serum and chick embryo extract give rise to large numbers of cells expressing immunoreactivity for substance P (SP), a neuropeptide found in sensory neurons. These cells arise from cycling precursors, but do not appear to divide after expressing SP. The SP-positive cells in cranial neural crest cultures express both neurofilament and the Q211 antigen, but those in trunk cultures express only the Q211 antigen. In both cranial and trunk cultures, large subpopulations of the SP-positive cells express tyrosine hydroxylase and/or choline acetyltransferase, neurotransmitter markers characteristic of autonomic neurons. This finding argues against the idea that SP expression necessarily indicates commitment to the sensory neuron lineage. I further show that embryonic dorsal root ganglion (DRG) cells retain the ability to coexpress SP and tyrosine hydroxylase in vitro, although to a lesser extent than do neural crest cells.     

Coexpression of sensory and autonomic neurotransmitter traits by avian neural crest cells in vitro

This study shows that explants of quail neural crest cultured in a medium containing serum and chick embryo extract give rise to large numbers of cells expressing immunoreactivity for substance P (SP), a neuropeptide found in sensory neurons. These cells arise from cycling precursors, but do not appear to divide after expressing SP. The SP-positive cells in cranial neural crest cultures express both neurofilament and the Q211 antigen, but those in trunk cultures express only the Q211 antigen. In both cranial and trunk cultures, large subpopulations of the SP-positive cells express tyrosine hydroxylase and/or choline acetyltransferase, neurotransmitter markers characteristic of autonomic neurons. This finding argues against the idea that SP expression necessarily indicates commitment to the sensory neuron lineage. I further show that embryonic dorsal root ganglion (DRG) cells retain the ability to coexpress SP and tyrosine hydroxylase in vitro although to a lesser extent than do neural crest cells.     

Prospero distinguishes sibling cell fate without asymmetric localization in the Drosophila adult external sense organ lineage

The adult external sense organ precursor (SOP) lineage is a model system for studying asymmetric cell division. Adult SOPs divide asymmetrically to produce IIa and IIb daughter cells; IIa generates the external socket (tormogen) and hair (trichogen) cells, while IIb generates the internal neuron and sheath (thecogen) cells. Here we investigate the expression and function of prospero in the adult SOP lineage. Although Prospero is asymmetrically localized in embryonic SOP lineage, this is not observed in the adult SOP lineage: Prospero is first detected in the IIb nucleus and, during IIb division, it is cytoplasmic and inherited by both neuron and sheath cells. Subsequently, Prospero is downregulated in the neuron but maintained in the sheath cell. Loss of prospero function leads to 'double bristle' sense organs (reflecting a IIb-to-IIa transformation) or 'single bristle' sense organs with abnormal neuronal differentiation (reflecting defective IIb development). Conversely, ectopic prospero expression results in duplicate neurons and sheath cells and a complete absence of hair/socket cells (reflecting a IIa-to-IIb transformation). We conclude that (1) despite the absence of asymmetric protein localization, prospero expression is restricted to the IIb cell but not its IIa sibling, (2) prospero promotes IIb cell fate and inhibits IIa cell fate, and (3) prospero is required for proper axon and dendrite morphology of the neuron derived from the IIb cell. Thus, prospero plays a fundamental role in establishing binary IIa/IIb sibling cell fates without being asymmetrically localized during SOP division. Finally, in contrast to previous studies, we find that the IIb cell divides prior to the IIa cell in the SOP lineage.     

Establishment of pure neuronal and muscle precursor cell cultures fromDrosophila early gastrula stage embryos

Summary Primary cultures ofDrosophila gastrula stage embryonic cells will divide and terminally differentiate into morphologically recognizable neurons and muscles. The phenotypically mixed nature of this primary culture system has made it difficult to effectively analyze various parameters of cell growth and differentiation for individual cell types. We report here a simple and economic method to separate early embryonic precursors for different cell types, using a shallow linear reorienting Ficoll gradient at unit gravity. The separated cells were collected into fractions, cultured, and analyzed for their growth and differentiation patterns. The larger and denser cells of the first fractions differentiated to yield pure neuronal cultures, as judged by morphologic, immunologic, and biochemical criteria. Cells in the last fractions differentiated into a predominantly muscle-enriched cell population, which also contained a very small percentage of neurons morphologically distinct from those in the pure neuronal fractions. Approximately 35% of the early gastrula stage embryonic cells differentiate into neuronal cells, and 65% of the non-neuronal lineage cells later develop into predominantly muscle population. The method is highly reproducible, can process 3×10⁷ cells per procedure, and the recovery is >90% of the input cells. The separated cells are suitable for cell biological analyses as well as for biochemical and molecular studies of neuron and muscle precursors. Deceased.     

Somatostatin-like immunoreactivity in the retinae of adult and embryonic chickens

Somatostatin, a tetradecapeptide that inhibits growth hormone release, has a widespread distribution in the central and peripheral nervous systems and other cell types. In the present investigation, the chicken neural retina was studied for the presence of structures exhibiting somatostatin-like immunoreactivity by utilizing an indirect immunofluorescence technique. Controls for specificity of staining were performed on alternate sections. Several types of distinctly labeled neurons and their processes were evident in sections of adult and late embryonic retinae. Cresyl violet staining showed that these neurons, which were scattered peripherally and more numerous centrally, occupied several strata within the inner nuclear, inner plexiform, and ganglion cell layers. Labeled neurites of immunoreactive perikarya coursed within these layers as well, often approaching other immunoreactive cells and fibers. The morphology and position of the somatostatin-containing neurons indicated that these neurons were amacrine, horizontal, or ganglion associational cells. These findings indicate that somatostatin is first detectable in the retina during the late embryonic stages of the chicken.     

BMP‐4 inhibits neural differentiation of murine embryonic stem cells

Members of the transforming growth factor‐β superfamily, including bone morphogenetic protein 4 (BMP‐4), have been implicated as regulators of neuronal and glial differentiation. To test for a possible role of BMP‐4 in early mammalian neural specification, we examined its effect on neurogenesis in aggregate cultures of mouse embryonic stem (ES) cells. Compared to control aggregates, in which up to 20% of the cells acquired immunoreactivity for the neuron‐specific antibody TuJ1, aggregates maintained for 8 days in serum‐free medium containing BMP‐4 generated 5‐ to 10‐fold fewer neurons. The action of BMP‐4 was dose dependent and restricted to the fifth through eighth day in suspension. In addition to the reduction in neurons, we observed that ES cell cultures exposed to BMP‐4 contained fewer cells that were immunoreactive for glial fibrillary acidic protein or the HNK‐1 neural antigen. Furthermore, under phase contrast, cultures prepared from BMP‐4–treated aggregates contained a significant proportion of nonneuronal cells with a characteristic flat, elongated morphology. These cells were immunoreactive for antibodies to the intermediate filament protein vimentin; they were rare or absent in control cultures. Treatment with BMP‐4 enhanced the expression of the early mesodermal genes brachyury and tbx6 but had relatively little effect on total cell number or cell death. Coapplication of the BMP‐4 antagonist noggin counteracted the effect of exogenous BMP‐4, but noggin alone had no effect on neuralization in either the absence or presence of retinoids. Collectively, our results suggest that BMP‐4 can overcome the neuralizing action of retinoic acid to enhance mesodermal differentiation of murine ES cells. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 271–287, 1999     

Mechanisms and molecules in motor neuron specification and axon pathfinding.

The vertebrate nervous system performs the most complex functions of any organ system. This feat is mediated by dedicated assemblies of neurons that must be precisely connected to one another and to peripheral tissues during embryonic development. Motor neurons, which innervate muscle and regulate autonomic functions, form an integral part of this neural circuitry. The first part of this review describes the remarkable progress in our understanding of motor neuron differentiation, which is arguably the best understood model of neuronal differentiation to date. During development, the coordinate actions of inductive signals from adjacent non-neural tissues initiate the differentiation of distinct motor neuron subclasses, with specific projection patterns, at stereotypical locations within the neural tube. Underlying this specialisation is the expression of specific homeodomain proteins, which act combinatorially to confer motor neurons with both their generic and subtype-specific properties. Ensuring that specific motor neuron subtypes innervate the correct target structure, however, requires precise motor axon guidance mechanisms. The second half of this review focuses on how distinct motor neuron subtypes pursue highly specific projection patterns by responding differentially to spatially discrete attractive and repulsive molecular cues. The tight link between motor neuron specification and axon pathfinding appears to be established by the dominant role of homeodomain proteins in dictating the ways that navigating motor axons interpret the plethora of guidance cues impinging on growth cones.