Identification of maxillary factor, a maxillary process-derived chemoattractant for developing trigeminal sensory axons

Trigeminal sensory axons project to several epithelial targets, including those of the maxillary and mandibular processes. Previous studies identified a chemoattractant activity, termed Maxillary Factor, secreted by these processes, which can attract developing trigeminal axons in vitro. We report that Maxillary Factor activity is composed of two neurotrophins, neurotrophin-3 (NT-3) and Brain-Derived Neurotrophic Factor (BDNF), which are produced by both target epithelium and pathway mesenchyme and which are therefore more likely to have a trophic effect on the neurons or their axons than to provide directional information, at least at initial stages of trigeminal axon growth. Consistent with this, the initial trajectories of trigeminal sensory axons are largely or completely normal in mice deficient in both BDNF and NT-3, indicating that other cues must be sufficient for the initial stages of trigeminal axon guidance.     

Mesenchyme formation from the trigeminal placodes of the mouse embryo

The trigeminal placode is a thickened region of ectodermal epithelium located along the side of the embryonic head. Mesenchyme escapes from the placode to form neurons of the trigeminal (V) ganglion. To further our knowledge of the morphogenesis of this escape, plastic thick sections were cut from mouse embryos and stained for light microscopy by using a technique which revealed escaping mesenchyme. The escape of trigeminal mesenchyme began at approximately 12 somites of age and was substantially complete by 30 somites. These results provided spatial/temporal orientation for a subsequent electron microscopic study. The first ultrastructural manifestation of escape was the penetration of an otherwise continuous basal lamina by small cell processes. The presence of longitudinally oriented microtubules within these processes suggests that mesenchymal cells escape through the basal lamina by using microtubules to direct/move their contents (e.g., the cell nucleus) into an enlarging process. Nuclei were distorted as they passed into these processes. This distortion suggests that basal lamina, together with a possible contribution from basal microfilaments, forms a rigid obstruction which is disrupted in the region from which a process is formed. In some cases a collar of basal lamina was observed around the necks of processes, but their distal membranes were invariably lamina-free. This lamina-free membrane is possibly that which is newly formed to accommodate the growing process. In later stages of escape, instances were observed in which the lamina was completely absent beneath an escaping cell and partially degraded beneath adjacent cells as well. These instances suggest that enzymatic digestion may play a role in degrading the lamina during mesenchymal escape. Apical desmosomes were often retained beyond the initial stages of escape. Mechanisms involved in their disruption are thus not among those which initiate escape.     

LIMK1 acts downstream of BMP signaling in developing retinal ganglion cell axons but not dendrites

The actin cytoskeleton inside extending axonal and dendritic processes must undergo continuous assembly and disassembly. Some extrinsic factors modulate actin turnover through controlling the activity of LIM kinase 1 (LIMK1), which phosphorylates and inactivates the actin depolymerizing factor cofilin. Here, we for the first time examine the function and regulation of LIMK1 in vivo in the vertebrate nervous system. Upon expression of wildtype or kinase-dead forms of the protein, dendrite growth by Xenopus retinal ganglion cells (RGCs) was unchanged. In contrast, maintaining a low, but significant level, of LIMK1 function in the RGC axon is critical for proper extension. Interestingly, bone morphogenetic protein receptor II (BMPRII) is a major regulator of LIMK1 in extending RGC axons, as expression of a BMPRII lacking the LIMK1 binding region caused a dramatic shortening of the axons. Previously, we found that BMPRIIs stimulate dendrite initiation in vivo. Thus, the fact that manipulation of LIMK1 activity failed to alter dendrite growth suggests that BMPs may activate distinct signalling pathways in axons and dendrites.     

Chondroitin sulphate-binding molecules may pattern central projections of sensory axons within the cranial mesenchyme of the developing mouse

During mammalian hindbrain development, sensory axons grow along highly stereotyped routes within the cranial mesenchyme to reach their appropriate entry points into the neuroepithelium. Thus, trigeminal ganglion axons always project to rhombomere (r)2, whilst facial/acoustic ganglia axons always project to r4. Axons are never observed to enter the mesenchyme adjacent to r3, raising the possibility that r3 mesenchyme contains an axon growth-inhibitory activity. Conversely, in mice which lack the erbB4 receptor (normally expressed in r3), trigeminal and facial/acoustic ganglia axons misproject into r3 mesenchyme, suggesting that the putative axon barrier is absent. To investigate this hypothesis, we have developed an in vitro model in which dissociated wild-type embryonic trigeminal ganglion neurons are cultured on longitudinal cryosections of embryonic mouse head. We observed that on wild-type embryonic day 10 (E10) cryosections, neurites generally failed to grow into r3 mesenchyme from the adjacent r2 or r4 mesenchyme. This barrier was removed if cryosections were pretreated with chondroitinase or were washed with excess chondroitin 6-sulphate or hypertonic saline. By contrast, when trigeminal neurons were seeded onto cryosections of E10 erbB4 -/- embryo heads their neurites readily entered mutant r3 mesenchyme. Immunohistochemical analysis demonstrated chondroitin-sulphated proteoglycans throughout the cranial mesenchyme in both wild-type and erbB4 -/- embryos. We propose that trigeminal axons are excluded from wild-type r3 mesenchyme by a growth-inhibitory activity which associates with chondroitin-sulphated proteoglycans and that the synthesis of this activity may rely on signals transduced by erbB receptors.     

Ganglion cells are required for normal progenitor- cell proliferation but not cell-fate determination or patterning in the developing mouse retina

The vertebrate retina develops from an amorphous sheet of dividing retinal progenitor cells (RPCs) through a sequential process that culminates in an exquisitely patterned neural tissue. A current model for retinal development posits that sequential cell-type differentiation is the result of changes in the intrinsic competence state of multipotent RPCs as they advance in time and that the intrinsic changes are influenced by continuous changes in the extracellular environment. Although several studies support the proposition that newly differentiated cells alter the extrinsic state of the developing retina, it is still far from clear what role they play in modifying the extracellular environment and in influencing the properties of RPCs. Here, we specifically ablate retinal ganglion cells (RGCs) as they differentiate, and we determine the impact of RGC absence on retinal development. We find that RGCs are not essential for changing the competence of RPCs, but they are necessary for maintaining sufficient numbers of RPCs by regulating cell proliferation via growth factors. Intrinsic rather than extrinsic factors are likely to play the critical roles in determining retinal cell fate.     

Quantitative morphological characteristics of developing neurons of the sensory trigeminal nuclei in kittens

Five types of neurons were distinguished in the sensory nuclei of the trigeminal nerve, stained by Golgi's method, in kittens aged 1–5 days and 30 days: reticular and short-dendritic cells (with few branches), and multipolar giant cells, arborescent, and bushy neurons (densely branching). Yet another special type of cell, with a few short dendrites and one long dendrite, was distinguished in preparations from the brain of newborn kittens. Analysis of the dimensions of the bodies, the number, length, and ramification of the dendrites, and the total ramification of the cell yielded quantitative morphological characteristics of these neurons at different times of development. These types of neurons differed in their qualitative and quantitative parameters and in the features of their maturation.Bushy neurons underwent regressive changes during development. Foci of maximal ramification of dendrites of densely branched neurons changed their location during the first months of life relative to the cell body, moving into the more distal regions of the dendrites. Differences in orientation of dendrites with foci of maximal ramification were found relative to neighboring brain formations, which depended on the types of cells and the animal's age. The high level of maturity of trigeminal neurons at birth was demonstrated.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Brain Institute, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 14, No. 6, pp. 592–600, November–December, 1982.     

Dendritic cells but not macrophages are targets for immune regulation by natural killer cells

Both dendritic cells (DC) and macrophages (M phi) stimulate lymphocyte proliferation in secondary mixed-lymphocyte (ML) reactions, though DC are approximately fourfold more effective. Natural killer (NK) cells suppress secondary ML reactions when DC are used, but NK cells do not suppress when M phi are used in these reactions. The findings are consistent with the idea that DC, but not M phi, are potential targets in immune regulation mediated by NK cells.     

Odorant representations are modulated by intra- but not interglomerular presynaptic inhibition of olfactory sensory neurons

Input to the central nervous system from olfactory sensory neurons (OSNs) is modulated presynaptically. We investigated the functional organization of this inhibition and its role in odor coding by imaging neurotransmitter release from OSNs in slices and in vivo in mice expressing synaptopHluorin, an optical indicator of vesicle exocytosis. Release from OSNs was strongly suppressed by heterosynaptic, intraglomerular inhibition. In contrast, inhibitory connections between glomeruli mediated only weak lateral inhibition of OSN inputs in slices and did not do so in response to odorant stimulation in vivo. Blocking presynaptic inhibition in vivo increased the amplitude of odorant-evoked input to glomeruli but had little effect on spatial patterns of glomerular input. Thus, intraglomerular inhibition limits the strength of olfactory input to the CNS, whereas interglomerular inhibition plays little or no role. This organization allows for control of input sensitivity while maintaining the spatial maps of glomerular activity thought to encode odorant identity.     

Retrograde but not anterograde bead movement in intact axons requires dynein

Dynein and kinesin have been implicated as the molecular motors that are responsible for the fast transport of axonal membranous organelles and vesicles. Experiments performed in vitro with partially reconstituted preparations have led to the hypothesis that kinesin moves organelles in the anterograde direction and dynein moves them in the retrograde direction. However, the molecular basis of transport directionality remains unclear. In the experiments described here, carboxylated fluorescent beads were injected into living Mauthner axons of lamprey and the beads were observed to move in both the anterograde and retrograde directions. The bead movement in both directions required intact microtubules, occurred at velocities approaching organelle fast transport in vivo, and was inhibited by vanadate at concentrations that inhibit organelle fast transport. When living axons were injected with micromolar concentrations of vanadate and irradiated at 365 nm prior to bead injections, a treatment that results in the V1 photolysis of dynein, the retrograde movement of the beads was specifically abolished. Neither the ultraviolet irradiation alone nor the vanadate alone produced the retrograde-specific inhibition. These results support the hypothesis that dynein is required for retrograde, but not anterograde, transport in vivo. © 1995 John Wiley & Sons, Inc.     

Correlation of acid phosphatase but not alkaline phosphatase activity with myelination in the developing mouse brain

• 1.1. Neonatal mice received subcutaneous injections of buffer, thiourea (TU) or propylthiouracil (PTU).
• 2.2. The PTU-treated mice were sacrificed on postnatal day 14 (P14) and the TU-treated mice on P28.
• 3.3. Brain weights of the TU- and PTU-treated mice were not significantly different from the controls.
• 4.4. Acid but not alkaline phosphatase activity in the braistem decreased after TU and PTU treatment.
• 5.5. Myelination as indicated by intensity of luxol fast blue staining was weaker in drug-treated animals.
• 6.6. The level of myelin marker enzyme, 2′,3′-cyclic nucleotide 3′-phosphohydrolase, was lower in the brainstem of PTU-treated animals.
• 7.7. The results suggest a correlation between acid phosphatase but not alkaline phosphatase activity with myelination in the developing mouse brain.

     

Synthesis of nerve growth factor mRNA in cultures of developing mouse whisker pad, a peripheral target tissue of sensory trigeminal neurons

The developmental increase in the level of NGF mRNA in mouse maxillary process/whisker pad is paralleled in vivo by the biochemical and morphological differentiation of whisker pad epidermis, i.e., changes in the keratin expression pattern and the appearance of hair follicles. In cultures of maxillary processes, however, depending on the age of explanted tissue, the increase in NGF mRNA levels either precedes or follows the appearance of epithelial differentiation markers. In addition, we found that prevention of epithelial differentiation by retinoic acid did not affect the increase in NGF mRNA levels. Only in explants from E11.5 embryos was the timing of NGF mRNA production comparable to that of the in vivo situation, whereas at earlier stages (E10/10.5) NGF mRNA levels increased slowly but never reached in vivo levels, even after extended culture periods. However, the amount of NGF mRNA in E10/10.5 maxillary processes was strongly increased in the presence of medium conditioned by E11.5 explants. This effect was not mimicked by the factors IL-1 beta and TGF-beta 1 known to induce NGF mRNA in other systems. It is concluded that the developmental increase in NGF mRNA levels in developing mouse whisker pad is not linked to epidermal differentiation. Interestingly, it is strongly stimulated by a soluble factor(s) produced within the tissue.     

Neurotrophin-independent attraction of growing sensory and motor axons towards developing Xenopus limb buds in vitro

The mechanisms for directing axons to their targets in developing limbs remain largely unknown though recent studies in mice have demonstrated the importance of neurotrophins in this process. We now report that in co-cultures of larval Xenopus laevis limb buds with spinal cords and dorsal root ganglia of Xenopus and axolotl (Ambystoma mexicanum) axons grow directly to the limb buds over distances of up to 800 microm and in particular to sheets of epidermal cells which migrate away from the limb buds and also tail segments in culture. This directed axonal growth persists in the presence of trk-IgG chimeras, which sequester neurotrophins, and k252a, which blocks their actions mediated via trk receptors. These findings indicate that developing limb buds in Xenopus release diffusible factors other than neurotrophins, able to attract growth of sensory and motor axons over long distances.     

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.     

Dorsally derived netrin 1 provides an inhibitory cue and elaborates the 'waiting period' for primary sensory axons in the developing spinal cord

Dorsal root ganglion (DRG) neurons extend axons to specific targets in the gray matter of the spinal cord. During development, DRG axons grow into the dorsolateral margin of the spinal cord and projection into the dorsal mantle layer occurs after a ;waiting period' of a few days. Netrin 1 is a long-range diffusible factor expressed in the ventral midline of the developing neural tube, and has chemoattractive and chemorepulsive effects on growing axons. Netrin 1 is also expressed in the dorsal spinal cord. However, the roles of dorsally derived netrin 1 remain totally unknown. Here, we show that dorsal netrin 1 controls the correct guidance of primary sensory axons. During the waiting period, netrin 1 is transiently expressed or upregulated in the dorsal spinal cord, and the absence of netrin 1 results in the aberrant projection of sensory axons, including both cutaneous and proprioceptive afferents, into the dorsal mantle layer. Netrin 1 derived from the dorsal spinal cord, but not the floor plate, is involved in the correct projection of DRG axons. Furthermore, netrin 1 suppresses axon outgrowth from DRG in vitro. Unc5c(rcm) mutant shows abnormal invasion of DRG axons as observed in netrin 1 mutants. These results are the first direct evidence that netrin 1 in the dorsal spinal cord acts as an inhibitory cue for primary sensory axons and is a crucial signal for the formation of sensory afferent neural networks.     

Demonstration of glutamate in primary sensory trigeminal neurons innervating dental pulp in rats]

Primary sensory trigeminal neurons supplying the dental pulp of incisors in rats were labelled by retrograde axonal transport. Using an auto-metallographic intensification procedure, 48 hrs. after injection of wheat-germ colloidal gold in the pulp, gold particles were detected in the cytoplasm of the neurons as black granulations. Glutamate was found in 45-60% of the neurons by submitting ganglion slices to an anti-glutamate immuno-serum revealed by immunocytochemistry. Among the neurons supplying the dental pulp of incisors by their peripheral process, 70% are Glu+, 30% Glu-. These observations suggest that the population of neurons supplying the dental pulp is not functionally homogeneous and that Glu- neurons use a different neurotransmitter. The coexistence of Glu+ and Glu- neurons could also indicate that glutamate expression is modulated during the life of these neurons.     

Quantitative morphological characteristics of developing neurons after partial deafferentation of the sensory trigeminal nuclei

We have studied different types of neurons in the sensory trigeminal nuclei stained by the Golgi method in kittens aged 30 days with bilateral transection of the lingual nerve, made on the fifth postnatal day. We have shown that deprivation of afferent inflow from the tongue to trigeminal neurons leads to changes in the structure of all types of cells: reticular, arborescent, and bushy (68, 61, and 46% neurons respectively had changed), short-dendrite cells having changed to a lesser extent (16% changed neurons). The multipolar giant neurons hardly changed. The structural changes involved changes in the size of the bodies, number, length, and ramification of dendrites, and changes in their orientation and pattern of ramification, compared to the normal. We observed destructive changes resulting in a decrease in the quantitative parameters, and constructive changes resulting in an increase in the latter. Reticular and arborescent neurons showed both destructive and constructive changes, the short-dendritic neurons mainly constructive changes, and bushy neurons mainly destructive changes. The analysis of the differently directed rearrangements of the dendrite geometry in different types of deafferentated trigeminal neurons allowed us to put forward some proposals concerning the different functional role of these groups of cells in the system of afferent impulsation entering via the lingual nerve.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR. Institute of the Brain of the All-Union Research Center of Mental Health, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 17, No. 4, pp. 522–530, July–August, 1985.