Roger J. Keynes

Isolation from chick somites of a glycoprotein fraction that causes collapse of dorsal root ganglion growth cones

The segmented pattern of peripheral spinal nerves in higher vertebrates is generated by interactions between nerve cells and somites. Neural crest cells, motor axons, and sensory axons grow exclusively through anterior-half sclerotome. In chick embryos, posterior cells bind the lectins peanut agglutinin (PNA) and Jacalin. When liposomes containing somite extracts are applied to cultures of chick sensory neurons, growth cones collapse abruptly, recovering within 4 hr of liposome removal. Collapse activity is eliminated by immobilized PNA, and SDS-PAGE demonstrates two major components (48K and 55K), which are absent from anterior-half sclerotome. Rabbit polyclonal antibodies against these components recognize only posterior cells and may also be used to eliminate collapse activity. We suggest that spinal nerve segmentation is produced by inhibitory interactions between these components and growth cones.

AXON GUIDANCE MOLECULES

To survey axon guidance in 1995 is to cover a very different field from some 30 years ago, when Sperry elaborated his idea of chemoaffinity to explain the precision of regeneration of the amphibian retinotectal map (Sperry, 1963). With hindsight we know that Sperry was overenthusiastic in his attribution of molecular labels to define tectal addresses and that the role of electrical activity in refining connectivity was yet to be fully appreciated. But at least he could envisage the problem in molecular terms and, in so doing, rekindled interest in a spectacular feat of biology. That interest has been growing ever since. The biology of axon guidance has been reviewed by Goodman and Shatz (1993). Here we concentrate on recent progress in understanding the nature and mechanism of axon guidance molecules, defining these as molecules outside the growth cone that assist in its selection of the correct pathway and target by changing its direction of growth. After synapse formation, connections may be refined and remodeled (address selection) by a process that is more dependent on patterns of electrical activity (Goodman and Shatz, 1993). Since guidance molecules can be both pathway derived and target derived, this article complements the accompanying review on target recognition (Garrityandzipursky, 1995 [this issueof Cc//j). The identification of growth cone receptors for guidance molecules is also critical, but is still at an early stage (see below). Lastly, understanding how growth cones turn also requires the elucidation of the signaling pathways within them that transduce receptor activation into changes in the dynamics and arrangement of the cytoskeleton (reviewed by Doherty and Walsh, 1994; see also Tanaka and Sabry, 1995 [this issue of Cc//j), and this will not be considered here. The possibility that guidance molecules exert either short-range (cell-cell) or long-range (diffusion gradient) influences on growth cones has been recognized for many years. More recently, it has become clear that guidance may be both attractive or repulsive (Goodman and Shatz, 1993; Keynes and Cook, 1995) and that these influences may be exerted over both short and long ranges by membersof the same molecular family. And as increasing numbers of candidates have been characterized, the earlier concept of guidance by differential adhesion has been modified by a realization that the guidance molecules, whether attractive, repulsive, short-range, or long-range, may direct growth cones via signal transduction mechanisms (see also Tanaka and Sabry, 1995). Attractive and Review

A critical role for sonic hedgehog signaling in the early expansion of the developing brain

The mechanisms that coordinate the three-dimensional shape of the vertebrate brain during development are largely unknown. We have found that sonic hedgehog (Shh) is crucial in driving the rapid, extensive expansion of the early vesicles of the developing midbrain and forebrain. Transient displacement of the notochord from the midbrain floor plate resulted in abnormal folding and overall collapse of the vesicles, accompanied by reduced cell proliferation and increased cell death in the midbrain. Simultaneously, expression of Shh decreased locally in the notochord and floor plate, whereas overt patterning and differentiation proceeded normally. Normal midbrain expansion was restored by implantation of Shh-secreting cells in a dose-dependent manner; conversely, expansion was retarded following antagonism of the Shh signaling pathway by cyclopamine. Our results indicate that Shh signaling from the ventral midline is essential for regulating brain morphogenesis during early development.

Regeneration of mouse peripheral nerves in degenerating skeletal muscle: guidance by residual muscle fibre basement membrane

The part played by basement membrane in the guidance of peripheral nerve growth in vivo has been assessed by examining the capacity of degenerating mouse muscle to support the regeneration of the cut sciatic and saphenous nerves. Ethanol and formaldehyde-fixed gluteus maximus muscles were implanted around the contralateral cut nerves. The subsequent nerve growth into the degenerating muscle was assessed by silver staining after 3, 4 and 10 days. By 4 days, linear axonal growth was seen, parallel to the length of the muscle fibres, and coinciding with the onset of degeneration of the sarcoplasm. Transverse sections of the 10 day preparations showed that over 90% of linearly growing axons were located inside the remaining sheaths of muscle fibre basement membrane. This relationship was confirmed by electron microscopy of ruthenium red-stained preparations. Both motor and sensory axons were able to grow in this manner, for electrophysiological testing revealed the presence of motor axons from the sciatic nerve, while the saphenous nerve contains only sensory axons. Identical growth was seen at 10 days in muscles caused to degenerate by incubation in distilled water. However, linear growth did not occur in live-innervated and glutaraldehyde-fixed muscles, in which muscle fibre architecture was preserved. It is concluded that basement membrane derived from muscle can promote peripheral nerve regeneration. Furthermore, both motor and sensory axons show a strong preference for growth along its inner surface, the basal lamina.

Neural crest patterning and the evolution of the jaw

Here we present ideas connecting the behaviour of the cranial neural crest during development with the venerable, perhaps incorrect, view that gill‐supporting cartilages of an ancient agnathan evolved into the skeleton of an early gnathostomes jaw. We discuss the pattern of migration of the cranial neural crest ectomesenchyme in zebrafish, along with the subsequent arrangement of postmigratory crest and head mesoderm in the nascent pharyngeal segments (branchiomeres), in diverse gnathostomes and in lampreys. These characteristics provide for a plausible von Baerian explanation for the problematic inside‐outside change in topology of the gills and their supports between these 2 major groups of vertebrates. We consider it likely that the jaw supports did indeed arise from branchiomeric cartilages.

The role of Schwann cells in the regeneration of peripheral nerve axons through muscle basal lamina grafts

Evacuated muscle is a possible substitute for nerve autografts in the repair of damaged peripheral nerves. Previous experiments have shown that killed or evacuated muscle grafts are as effective as nerve autografts for bridging gaps of up to 4 cm between proximal and distal nerve stumps. Evacuated muscle grafts are made of extracellular matrix components, which are good substrates for axon growth in vitro. However, experiments in vivo have generally demonstrated that live Schwann cells are essential for successful axon regeneration. In the present experiments we have used immunohistochemical techniques with anti-S100 and anti-neurofilament antibodies to visualize axon growth and Schwann cell migration into muscle grafts over the first 10 days following grafting. We only saw axons growing into grafts accompanied by Schwann cells, and most though not all Schwann cells were associated with axons. Schwann cell migration from the proximal stump in association with axons was much faster and more extensive than from the distal stump. We examined muscle grafts over the first 20 days after grafting by electron microscopy. Regenerating axons were always associated with Schwann cells, which were mostly in the basal lamina-lined tubes left by the evacuated myofibrils. A comparison between evacuated muscle grafts and grafts in which the muscle had been killed but not evacuated revealed that 7 days after grafting there were more than twice as many regenerated axons in and distal to the evacuated grafts, but that by 20 days the numbers of axons were similar in the two groups.

Repulsive and inhibitory signals

Repulsive or inhibitory interactions between growth cones and their environment are now widely implicated in neural development and regeneration. Over the past year, descriptive studies of the various neuronal systems in which repulsion may participate have clarified its biology. Molecular and genetic studies have also provided the necessary entry point for further experimental manipulations, and are beginning to yield important clues regarding the function of repulsion in vivo. Although candidate second messengers underlying the growth cone response have been identified, they have yet to be incorporated into a comprehensive mechanism.

An assessment of the spread of the signal for terminal sprouting within and between muscles.

Certain muscles of the mouse and rat have been studied in order to assess how far a signal from denervated muscle can spread to elicit terminal sprouting from intact endplates. Denervation of the muscles surrounding the rat foot 4th lumbrical muscle caused no terminal sprouting in the 4th lumbrical itself. In the hemidenervated mouse gluteus maximus terminal sprouting was restricted to the central region of the muscle where innervated and denervated fibres intermingle. There was no enhancement of such sprouting if the underlying and closely apposed gluteus medius was simultaneously denervated. Hemidenervation of the mouse diaphragm and interscutularis, where intact endplates lie near to denervated muscle fibres, produced no terminal sprouting. Hemidenervation of the mouse platysma, where intact endplates often lie adjacent to denervated muscle fibres, similarly produced no significant response. However, all muscles were capable of producing extensive terminal sprouting in response to paralysis induced by botulinum toxin. The stimulus for terminal sprouting produced by an inactive muscle fibre must therefore be effective only on the fibres own terminal or immediately adjacent terminals.

An analysis of astrocytic cell lines with different abilities to promote axon growth

The adult mammalian central nervous system (CNS) lacks the capacity to support axonal regeneration. There is increasing evidence to suggest that astrocytes, the major glial population in the CNS, may possess both axon-growth promoting and axon-growth inhibitory properties and the latter may contribute to the poor regenerative capacity of the CNS. In order to examine the molecular differences between axon-growth permissive and axon-growth inhibitory astrocytes, a panel of astrocyte cell lines exhibiting a range of axon-growth promoting properties was generated and analysed. No clear correlation was found between the axon-growth promoting properties of these astrocyte cell lines with: (i) the expression of known neurite-outgrowth promoting molecules such as laminin, fibronectin and N-cadherin; (ii) the expression of known inhibitory molecules such tenascin and chondroitin sulphate proteoglycan; (iii) plasminogen activator and plasminogen activator inhibitor activity; and (iv) growth cone collapsing activity. EM studies on aggregates formed from astrocyte cell lines, however, revealed the presence of an abundance of extracellular matrix material associated with the more inhibitory astrocyte cell lines. When matrix deposited by astrocyte cell lines was assessed for axon-growth promoting activity, matrix from permissive lines was found to be a good substrate, whereas matrix from the inhibitory astrocyte lines was a poor substrate for neuritic growth. Our findings, taken together, suggest that the functional differences between the permissive and the inhibitory astrocyte cell lines reside largely with the ECM.

Tsukushi Functions as an Organizer Inducer by Inhibition of BMP Activity in Cooperation with Chordin

During chick gastrulation, inhibition of BMP signaling is required for primitive streak formation and induction of Hensens node. We have identified a unique secreted protein, Tsukushi (TSK), which belongs to the Small Leucine-Rich Proteoglycan (SLRP) family and is expressed in the primitive streak and Hensens node. Grafts of cells expressing TSK in combination with the middle primitive streak induce an ectopic Hensens node, while electroporation of TSK siRNA inhibits induction of the node. In Xenopus embryos, TSK can block BMP function and induce a secondary dorsal axis, while it can dorsalize ventral mesoderm and induce neural tissue in embryonic explants. Biochemical analysis shows that TSK binds directly to both BMP and chordin and forms a ternary complex with them. These observations indicate that TSK is an essential dorsalizing factor involved in the induction of Hensens node.