Michael J. Bastiani

Guidance of pioneer growth cones: Filopodial contacts and coupling revealed with an antibody to Lucifer Yellow

Abstract We are interested in the factors that guide individual neuronal growth cones during embryonic development. We have developed an antibody to the fluorescent dye Lucifer Yellow. We use the antibody here to examine the specific filopodial contacts and dye coupling by the first growth cones in the grasshopper embryo that navigate in an axonless environment. We have studied the distribution and apparent selective adhesion of the filopodia from these pioneering growth cones in the central nervous system and periphery. Our results suggest that selective filopodial adhesion to specific “landmark” cells may play an important role in the guidance of pioneer growth cones.

Neuronal specificity and growth cone guidance in grasshopper and Drosophila embryos

Abstract Studies on a variety of vertebrate and invertebrate nervous systems point to the existence of specific chemical cues for both growth cone guidance and target recognition. Open questions, however, include how many recognition labels and how specific is their expression? Here we review our recent studies on insect embryos aimed at trying to answer these questions, focusing in particular on the specificity of two identified growth cones (aCC and pCC) in the CNS of the grasshopper and Drosophila embryos. Ablation experiments demonstrate the selective affinities of the aCC and pCC growth cones for particular axonal surfaces; in some cases such ablations result in temporal and spatial transplants which further demonstrate this exquisite specificity. Our results suggest the differential expression of many different surface recognition molecules in the developing CNS (labelled pathways hypothesis), a notion reminiscent of Sperrys chemoaffinity hypothesis. The SOX2 monoclonal antibody reveals an antigen in the Drosophila embryo that correlates with this model: SOX2 is expressed by the aCC and the small subset of neurons whose axons fasciculate with it.

Axon Regeneration Genes Identified by RNAi Screening in C. elegans

Axons of the mammalian CNS lose the ability to regenerate soon after development due to both an inhibitory CNS environment and the loss of cell-intrinsic factors necessary for regeneration. The complex molecular events required for robust regeneration of mature neurons are not fully understood, particularly in vivo. To identify genes affecting axon regeneration in Caenorhabditis elegans, we performed both an RNAi-based screen for defective motor axon regeneration in unc-70/β-spectrin mutants and a candidate gene screen. From these screens, we identified at least 50 conserved genes with growth-promoting or growth-inhibiting functions. Through our analysis of mutants, we shed new light on certain aspects of regeneration, including the role of β-spectrin and membrane dynamics, the antagonistic activity of MAP kinase signaling pathways, and the role of stress in promoting axon regeneration. Many gene candidates had not previously been associated with axon regeneration and implicate new pathways of interest for therapeutic intervention.

The growth factor SVH-1 regulates axon regeneration in C. elegans via the JNK MAPK cascade

The ability of neurons to undergo regenerative growth after injury is governed by cell-intrinsic and cell-extrinsic regeneration pathways. These pathways represent potential targets for therapies to enhance regeneration. However, the signaling pathways that orchestrate axon regeneration are not well understood. In Caenorhabditis elegans, the Jun N-terminal kinase (JNK) and p38 MAP kinase (MAPK) pathways are important for axon regeneration. We found that the C. elegans SVH-1 growth factor and its receptor, SVH-2 tyrosine kinase, regulate axon regeneration. Loss of SVH-1–SVH-2 signaling resulted in a substantial defect in the ability of neurons to regenerate, whereas its activation improved regeneration. Furthermore, SVH-1–SVH-2 signaling was initiated extrinsically by a pair of sensory neurons and functioned upstream of the JNK-MAPK pathway. Thus, SVH-1–SVH-2 signaling via activation of the MAPK pathway acts to coordinate neuron regeneration response after axon injury.

Endocannabinoid-Goα signalling inhibits axon regeneration in Caenorhabditis elegans by antagonizing Gqα-PKC-JNK signalling

The ability of neurons to regenerate their axons after injury is determined by a balance between cellular pathways that promote and those that inhibit regeneration. In Caenorhabditis elegans, axon regeneration is positively regulated by the c-Jun N-terminal kinase mitogen activated protein kinase pathway, which is activated by growth factor-receptor tyrosine kinase signalling. Here we show that fatty acid amide hydrolase-1, an enzyme involved in the degradation of the endocannabinoid anandamide (arachidonoyl ethanolamide), regulates the axon regeneration response of γ-aminobutyric acid neurons after laser axotomy. Exogenous arachidonoyl ethanolamide inhibits axon regeneration via the Goα subunit GOA-1, which antagonizes the Gqα subunit EGL-30. We further demonstrate that protein kinase C functions downstream of Gqα and activates the MLK-1-MEK-1-KGB-1 c-Jun N-terminal kinase pathway by phosphorylating MLK-1. Our results show that arachidonoyl ethanolamide induction of a G protein signal transduction pathway has a role in the inhibition of post-development axon regeneration.

The First Growth Cones in the Central Nervous System of the Grasshopper Embryo

What factors guide the advancing tips of individual growth cones during embryonic development? One way to answer this question is to examine and manipulate a developing embryo in which the growth cones of different neurons are confronted with the same environment and yet make different and stereotyped choices of which way to grow. The best such divergent choice to study would be one in which the individual growth cones are identifiable and highly accessible and their environment is stereotyped and relatively simple. We have been studying several different examples in the grasshopper embryo that meet these criteria: identified neurons with growth cones that make divergent choices (e.g., Goodman et al., 1982;Taghert et al., 1982; Raper et al., 1983a,b). In the example described in this chapter, the growth cones make divergent choices very early in embryogenesis when the terrain is relatively simple, the distances short, and the number of possible cells involved small. We will discuss the very first growth cones and the very first axonal pathways in the central nervous system (CNS) of the grasshopper embryo.

Molecular interactions of the neuronal GPI-anchored lipocalin Lazarillo

Lazarillo, a glycoprotein involved in axon growth and guidance in the grasshopper embryo, is the only member of the lipocalin family that is attached to the cell surface by a GPI anchor. Recently, the study of Lazarillo homologous genes in Drosophila and mouse has revealed new functions in the regulation of lifespan, stress resistance and neurodegeneration. Here we report an analysis of biochemical properties of Lazarillo to gain insight into the molecular basis of its physiological function. Recombinant forms of the grasshopper protein were expressed in two different systems to test: (1) potential binding of several hydrophobic ligands; (2) protein–protein homophilic interactions; and (3) whether interaction with the function‐blocking mAb 10E6 interferes with ligand binding. We tested 10 candidate ligands (retinoic acid, heme, bilirubin, biliverdin, ecdysterone, juvenile hormone, farnesol, arachidonic acid, linoleic acid and palmitic acid), and monitored binding using electrophoretic mobility shift, absorbance spectrum, and fluorimetry assays. Our work indicates binding to heme and retinoic acid, resulting in increased electrophoretic mobility, as well as to fatty acids, resulting in multimerization. Retinoic acid and fatty acids binding were confirmed by fluorescence titration, and heme binding was confirmed with absorbance spectrum assays. We demonstrate that Lazarillo oligomerizes in solution and can form clusters in the plasma membrane when expressed and GPI‐anchored to the cell surface, however it is unable to mediate cell–cell adhesion. Finally, by ligand‐mAb competition experiments we show that ligand‐binding alone cannot be the key factor for Lazarillo to perform its function during axonal growth in the grasshopper embryo. Copyright

The First Neuronal Growth Cones in Insect Embryos

Little is known about the mechanisms that generate neuronal specificity during development. Unraveling these mechanisms will require an understanding at the cellular and molecular level of how individual neurons recognize and interact with one another during development. Whereas mammalian nervous systems have an enormous number of neurons, the simpler nervous systems of invertebrates with their relatively small number of identified neurons can provide excellent model systems for studying neuronal specificity.

Growth Cone Guidance and Cell Recognition in Insect Embryos

In contrast to the complex central nervous system of most vertebrates, the insect CNS is relatively simple. The grasshopper CNS, for example, consists of a brain and a chain of segmental ganglia, each of which contains about 1000 neurons. Most of these neurons can be individually identified according to their unique axonal and dendritic morphology and their unique pattern of synaptic connections. This notion of unique identified neurons first arose dur ing the nineteenth century with descriptions of giant axons, which were repeatedly located in particular regions of the nerve cord. With the advent of intracellular dye-injection techniques (e.g., Stretton and Kravitz, 1968; Pitman et al., 1972; Stewart, 1978), many neurons became individually identified according to the location of their axons and dendrites in the neuropil and connectives. The spatial relationship of these processes to one another within the neuropil was then explored (e.g.,Tyrer and Gregory, 1982); it was shown that the axons and dendrites of identified neurons run in particular regions of specific tracts and commissures.

Heterochronic Genes Turn Back the Clock in Old Neurons

A signaling pathway is implicated in the age-dependent decline of neuron regeneration. [Also see Report by Zou et al.] Although some neuron types regenerate better than others, all neurons lose the ability to regenerate with age. This intrinsic decline is the primary cause of regeneration failure even in permissive environments. This was shown in 1995 by comparing the regeneration ability of retinal neurons from different aged retinas growing into tectums of different ages (1). Embryonic retinal axons regrew into tectum of any age, including older tectum with an inhibitory glial environment, whereas postnatal day 2 or older retinal axons failed to regrow even into embryonic tectum. This indicated a “programmed” loss of axon regeneration ability with neuron age. Similarly, young hindbrain neurons transplanted into older spinal cords could regenerate axons into a normally inhibitory myelinated environment (2). Despite the clear therapeutic implications of these observations, the underlying molecular mechanisms controlling age-dependent regenerative capacity were unclear. On page 372 in this issue, Zou et al. (3) report that the highly conserved let-7–LIN-41 heterochronic signaling pathway is responsible for part of the age-related decline in axon regeneration in the worm Caenorhabditis elegans.