PiggyBac transgenic strategies in the developing chicken spinal cord

The chicken spinal cord is an excellent model for the study of early neural development in vertebrates. However, the lack of robust, stable and versatile transgenic methods has limited the usefulness of chick embryos for the study of later neurodevelopmental events. Here we describe a new transgenic approach utilizing the PiggyBac (PB) transposon to facilitate analysis of late-stage neural development such as axon targeting and synaptic connection in the chicken embryo. Using PB transgenic approaches we achieved temporal and spatial regulation of transgene expression and performed stable RNA interference (RNAi). With these new capabilities, we mapped axon projection patterns of V2b subset of spinal interneurons and visualized maturation of the neuromuscular junction (NMJ). Furthermore, PB-mediated RNAi in the chick recapitulated the phenotype of loss of agrin function in the mouse NMJ. The simplicity and versatility of PB-mediated transgenic strategies hold great promise for large-scale genetic analysis of neuronal connectivity in the chick.     

Type III neuregulin 1 regulates pathfinding of sensory axons in the developing spinal cord and periphery

Sensory axons must develop appropriate connections with both central and peripheral targets. Whereas the peripheral cues have provided a classic model for neuron survival and guidance, less is known about the central cues or the coordination of central and peripheral connectivity. Here we find that type III Nrg1, in addition to its known effect on neuron survival, regulates axon pathfinding. In type III Nrg1(-/-) mice, death of TrkA(+) nociceptive/thermoreceptive neurons was increased, and could be rescued by Bax elimination. In the Bax and type III Nrg1 double mutants, axon pathfinding abnormalities were seen for TrkA(+) neurons both in cutaneous peripheral targets and in spinal cord central targets. Axon guidance phenotypes in the spinal cord included penetration of axons into ventral regions from which they would normally be repelled by Sema3A. Accordingly, sensory neurons from type III Nrg1(-/-) mice were unresponsive to the repellent effects of Sema3A in vitro, which might account, at least in part, for the central projection phenotype, and demonstrates an effect of type III Nrg1 on guidance cue responsiveness in neurons. Moreover, stimulation of type III Nrg1 back-signaling in cultured sensory neurons was found to regulate axonal levels of the Sema3A receptor neuropilin 1. These results reveal a molecular mechanism whereby type III Nrg1 signaling can regulate the responsiveness of neurons to a guidance cue, and show that type III Nrg1 is required for normal sensory neuron survival and axon pathfinding in both central and peripheral targets.     

Morphology of early developing oligodendrocytes in the ventrolateral spinal cord of the chicken

The oligodendroglial population includes Type I and II cells related to several thin axons, Type III cells with a few processes in relation to relatively thick axons and Type IV cells related to a single thick axon. This structural diversity of oligodendrocytes is accompanied by a molecular heterogeneity. In the chicken spinal cord, oligodendrocytes have begun to contact axons at embryonic day (E)10 and compact sheaths have appeared by E12. At the latter stage, most sheath-forming oligodendrocytes contact more than one axon. At E15, however, each sheath-forming cell seems to have developed a Schwann cell-like anatomy, being related to a single axon. Based on these findings, the present study examines more thoroughly the anatomy of early developing oligodendrocytes in the chicken spinal cord. Examination of slices immunostained with antibodies against the oligodendroglial marker O4 showed that a few positive cells are present at E6, after which the occurrence increases with age. At E12 most immunostained cells have two or more processes. At E15 however, dye-injected oligodendrocytes have developed a Type IV structure. Between E12 and E15, mean sheath length increases about 4×, from 50 μm to over 200 μm, while the length of the spinal cord increases 36% only. This indicates that early oligodendrocytes in chicken white matter develop a Type IV anatomy between E12 and E15 through an elimination of sheaths.     

Specification of synaptic connections between sensory and motor neurons in the developing spinal cord

Experimental studies of mechanisms underlying the specification of synaptic connections in the monosynaptic stretch reflex of frogs and chicks are described. Sensory neurons innervating the triceps brachii muscles of bullfrogs are born throughout the period of sensory neurogenesis and do not appear to be related clonally. Instead, the peripheral targets of these sensory neurons play a major role in determining their central connections with motoneurons. Developing thoracic sensory neurons made to project to novel targets in the forelimb project into the brachial spinal cord, which they normally never do. Moreover, these foreign sensory neurons make monosynaptic excitatory connections with the now functionally appropriate brachial motoneurons. Normal patterns of neuronal activity are not necessary for the formation of specific central connections. Neuromuscular blockade of developing chick embryos with curare during the period of synaptogenesis still results in the formation of correct sensory-motor connections. Competitive interactions among the afferent fibers also do not seem to be important in this process. When the number of sensory neurons projecting to the forelimb is drastically reduced during development, each afferent still makes central connections of the same strength and specificity as normal. These results are discussed with reference to the development of retinal ganglion cells and their projections to the brain. Although many aspects of the two systems are similar, patterned neural activity appears to play a much more important role in the development of the visual pathway than in the spinal reflex pathway described here.     

PlexinA1 signaling directs the segregation of proprioceptive sensory axons in the developing spinal cord

As different classes of sensory neurons project into the CNS, their axons segregate and establish distinct trajectories and target zones. One striking instance of axonal segregation is the projection of sensory neurons into the spinal cord, where proprioceptive axons avoid the superficial dorsal horn-the target zone of many cutaneous afferent fibers. PlexinA1 is a proprioceptive sensory axon-specific receptor for sema6C and sema6D, which are expressed in a dynamic pattern in the dorsal horn. The loss of plexinA1 signaling causes the shafts of proprioceptive axons to invade the superficial dorsal horn, disrupting the organization of cutaneous afferents. This disruptive influence appears to involve the intermediary action of oligodendrocytes, which accompany displaced proprioceptive axon shafts into the dorsal horn. Our findings reveal a dedicated program of axonal shaft positioning in the mammalian CNS and establish a role for plexinA1-mediated axonal exclusion in organizing the projection pattern of spinal sensory afferents.     

A brachial glycogen body in the spinal cord of the domestic chicken.

When cervical segments 14 to 15 of the chicken spinal cord are cut transversely and studied by routine histological and histochemical methods, an onion-shaped region, filled with thread-like fibers, is seen to surround the ependymal cells of the central canal and to be bounded laterally by the neural elements of the spinal gray matter. This area is negative for succinic dehydrogenase, beta-hydroxybutyrate dehydrogenase and cholinesterase activity, but very strongly periodic acid-Schiff positive. Diastase controls show the positive material to be glycogen. Parasagittal sections through this cervical region and into the upper thoracic cord, show the glycogen-rich region to extend longitudinally throughout the region. Because of its location and histochemical characterization, which are similar to that of the ventral portion of the glycogen body, the term brachial glycogen body is proposed for this structure.     

Bioelectric activity is required for regional specificity of sensory ganglion projections to spinal cord explants cultured in vitro

Summary Chronic blockade of spontaneous nerve impulses by means of tetrodotoxin leads to abnormally diffuse afferent projections into spinal cord cross-sections cultured for two to six weeks in vitro. In addition, even untreated explants which show a low level of spontaneous cord discharges failed to develop the normal degree of dorsal pathway selectivity. It is therefore concluded that centrally generated neuronal activity may play an important role in eliminating exuberant connections which, during early development, are transiently present in this part of the nervous system.     

Intermediate filament proteins in the developing chick spinal cord

The distribution of different intermediate filament (IF) proteins in the embryonic chick spinal cord was examined at several stages of development using immunohistochemical techniques, analytic gel electrophoresis, and electron microscopy. We have found that: (1) the fibroblast-type IF protein (vimentin) is present in virtually all of the replicating neuroepithelial cells of the early neural tube, as well as in radial glia, astrocytes, and Schwann cells in later stages of development; (2) the fibroblast-type IF protein is not detectable in definitive neurons; (3) the neurofilament proteins are first detectable in postmitotic neuroblasts at about the time of initial axon formation and they are restricted to neurons; (4) the astrocyte-type IF protein (glial fibrillary acidic protein) is in definitive astrocytes, but not in radial glia; (5) the prekeratin proteins are restricted to cells of the leptomeninges; and (6) the muscle-type IF protein (desmin) is restricted to vascular tissue in and around the developing spinal cord. These findings suggest that the fibroblast-type IF protein is the only IF protein in the early neuroepithelial cells and that the progeny of these cells will follow one of three different patterns of IF protein expression: (1) continued expression of only the fibroblast-type IF protein (radial glia); (2) expression of both the fibroblast-type IF protein and the astrocyte-type IF protein (astrocytes); or (3) expression of only the neurofilament proteins (neurons).     

VEGF modulates synaptic activity in the developing spinal cord

Although it has been documented that the nervous and the vascular systems share numerous analogies and are closely intermingled during development and pathological processes, interactions between the two systems are still poorly described. In this study, we investigated whether vascular endothelial growth factor (VEGF), which is a key regulator of vascular development, also modulates neuronal developmental processes. We report that VEGF enhances the gamma‐aminobutyric acid (GABA)/glycinergic but not glutamatergic synaptic activity in embryonic spinal motoneurons (MNs), without affecting MNs excitability. In response to VEGF, the frequency of these synaptic events but not their amplitude was increased. Blocking endogenous VEGF led to an opposite effect by decreasing frequency of synaptic events. We found that this effect occurred specifically at early developmental stages (E13.5 and E15.5) and vanished at the prenatal stage E17.5. Furthermore, VEGF was able to increase vesicular inhibitory amino acid transporter density at the MN membrane. Inhibition of single VEGF receptors did not modify electrophysiological parameters indicating receptor combinations or an alternative pathway. Altogether, our findings identify VEGF as a modulator of the neuronal activity during synapse formation and highlight a new ontogenic role for this angiogenic factor in the nervous system. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1110–1122, 2014     

Distinct neural precursors in the developing human spinal cord

Both embryonic and adult central nervous system have been shown to contain multipotent neural stem cells, but it is not yet clear whether they consist of a single or distinct populations of neural precursors. Since embryonic human neural precursors, particularly in the spinal cord, have not been extensively characterized, we have studied their behaviour at different days of gestation and in different culture conditions. Depending on dissociation and culture conditions, neurospheres which contain nestin- and vimentin-positive or only vimentin-positive neural precursors can be isolated. Whereas the former can be isolated only at early developmental stages, the latter appear to be present at all the stages examined, between 45 and 89 days of gestation. Furthermore, comparison of the effect of FGF, EGF and the two factors in combination on colony formation shows an additive effect of the two growth factors, indicating the existence of more than one type of neural precursor. Overall our results suggest that the human spinal cord contains distinct and dynamic populations of neural precursors which are developmentally regulated.     

2',3'-Cyclic nucleotide 3'-phosphohydrolase in the developing chick brain and spinal cord

Developmental changes of the 2',3'-cyclic nucleotide 3'-phosphohydrolase activity in the chick brain and spinal cord are reported. The greater part of increase in enzyme activity occurred between 18 days of incubation and 3 days after hatching in the whole brain, and between 18 and 21 days of incubation in the spinal cord. These periods are those of active myelination in the chick brain and spinal cord, respectively. The possibility was emphasized that 2',3'-cyclic nucleotide 3'-phosphohydrolase can be used as a marker for the myelin sheaths in the developing central nervous system. Comparisons were also made among the developmental changes in the forebrain, midbrain, brain stem, cerebellum, and spinal cord.