Jeffrey L. Denburg

A role for proteoglycans in the guidance of a subset of pioneer axons in cultured embryos of the cockroach

Several molecules involved in the development of the nervous system have specific binding sites for the glycosaminoglycan (GAG) side chains of proteoglycans. Exogenous GAGs should bind to these sites, competitively inhibit interactions with proteoglycans, and perturb development. GAGs added to the culture medium perturb the in situ growth of pioneer axons in cultured cockroach embryos by producing axon defasciculation and growth in incorrect directions. The specificity of this phenomenon is evident from the following observations: Of all the GAGs tested only heparin and heparan sulfate produced perturbation; of the six axon tracts being pioneered during the culture period only two of them are perturbed by the GAGs; and similar perturbations are produced when embryos are cultured in the presence of heparinase II and heparitinase.

Developmental stage-specific antigens in the nervous system of the cockroach

A specific effort was made to obtain monoclonal antibodies that bind to macromolecules that play a role in the development of the nervous system. It was considered that good candidates for such molecules were those that were only transiently present in the embryonic nervous system. Hybridomas were prepared from spleen cells taken from mice that had been immunized with nerve cords from cockroach embryos at the 43-50% stage of development. The hybridoma supernatants were screened for antibody binding to frozen sections of both embryonic and adult thoracic ganglia. Cell lines that produced monoclonal antibodies that transiently bound to the embryonic nervous system were saved and cloned. These developmental stage-specific monoclonal antibodies either did not bind to the adult nervous system or bound to it with a pattern very different from that in the embryonic nervous system. The developmental stage-specific antigens detected by these monoclonal antibodies were organized into four categories based on the part of the embryonic nervous system in which they were transiently localized. These include binding to the cell bodies of all neurons, cell bodies of subsets of neurons or neuroblasts, subsets of axons, and the neuropile. Preliminary biochemical characterization of the antigens showed that many of these antibodies were recognizing carbohydrate epitopes. Functions for these antigens, most of which are components of the cell surface, are tentatively proposed.

Elimination of inappropriate axonal branches of regenerating cockroach motor neurons as detected by the retrograde transport of horseradish peroxidase conjugated wheat germ agglutinin.

A procedure is described for identifying the motor neurons innervating individual cockroach muscles by use of the retrograde transport of extracellularly injected horseradish peroxidase and the staining of cells in whole mount preparations. A modification of this procedure using wheat germ agglutinin covalently conjugated to horseradish peroxidase is required in order to identify regenerating motor neurons that have an axonal process that had regrown into a particular leg muscle at various times after axotomy. At early stages of regeneration 23 out of 31 axotomized motor neurons with axons leaving the metathoracic ganglion through nerve root 5 send an axonal process into muscle 178. In intact animals this muscle receives innervation from only one motor neuron, Df. At later stages of regeneration these inappropriate axonal branches become eliminated until this muscle is again innervated only by Df. These results indicate that the specificity required for the reformation of the original innervation pattern of the leg muscles does not come from factors which are components of paths along the nerve root or which are being released from denervated muscles which could selectively control the direction of growth of axons of particular motor neurons.

Pattern formation during insect leg segmentation: Studies with a prepattern of a cell surface antigen

SummaryA monoclonal antibody (MAb) that binds to a cell surface antigen selectively localized to epithelial cells undergoing morphogenesis was used to study the segmentation of the growing embryonic leg of the cockroachPeriplaneta americana. The MAb labels circumferential stripes of cells at locations where invagination will occur to form the leg segments. The formation of these stripes precedes any morphological change in the epithelial layer or in individual cells. The temporal and spatial distribution of the antigen indicates the existence of a prepattern for leg segmentation, examination of which can give information about pattern generating mechanisms. Although highly stereotyped, the sequence in which the stripes appear does not follow a simple pattern proceeding in one direction along the proximal-distal axis. It is proposed that each stripe is a boundary in a positional field. Stripe formation leads to the division of the leg into a repeating series of identical positional fields. Three different mechanisms for the formation of stripes of MAb labeled cells have been observed and the role of each in the evolution of the insect leg is discussed. Measurements of leg and leg segment lengths when the various stripes appear has demonstrated considerable variation, particularly at the early stages of segmentation. Rules or mechanisms generating pattern at early stages of development are not rigid. Variations arising are compensated for by later occurring events so that stereotyped structures are formed.

An axon growth associated antigen is also an early marker of neuronal determination.

We have previously described the generation of a monoclonal antibody (DSS-3) that binds to all neurons in cockroach embryos at 50% development and to only a small subset of interneurons in the adult nervous system. This developmental stage-specific antigen was observed to reappear in all axotomized adult neurons that were undergoing axonal regeneration. In the present study the time course of the appearance of this growth-associated antigen during embryonic development was determined. Unexpectedly, the antigen was observed to be present in embryonic neurons long before axon growth. In addition, all cells in the CNS neuronal lineage (neuroblasts, ganglion mother cells, and neurons) bind the antibody as soon as they can be morphologically identified. However, the antigen is also transiently present in all neuroepithelial cells at a stage prior to the morphological differentiation of some of them to neuroblasts. Analogous patterns of DSS-3 binding to cells involved in the development of sensory neurons and leg pioneer neurons are observed. The DSS-3 antigen is therefore a very early marker for the capacity of ectodermal epithelial cells to develop along a neuronal lineage.

A molecular marker for epithelial morphogenesis in the cockroach

SummaryA molecular marker has been identified in embryos of the cockroach, Periplaneta americana, that is localized among epithelial cells to those directly involved in morphogenesis. A monoclonal antibody has been developed that selectively binds to epithelial cells undergoing any of three very different morphogenetic movements-invagination, evagination or epiboly. Neighboring cells not involved in these developmental processes are not labeled by the antibody. The antigen is transiently present on the cells for a period just prior to and during the morphogenetic activity. It is localized on the apical surface of the cells. The spatial, temporal and subcellular distributions of antibody binding during development indicate a role for the antigen in epithelial morphogenesis different from that of any previously described molecule.

Lectin receptors in the cockroach neuromuscular system. I. Distribution among the set of coxal depressor muscles

Fluorescent derivatives of plant lectins have been used to determine if there are biochemical differences among the cell surfaces of 6 muscles in the leg of the cockroach innervated in a fixed pattern by two identified motor neurons, Df or Ds. Histochemical analysis of the binding of fluorescein isothiocyanate conjugates of the lectins to frozen sections of muscles has demonstrated that the surfaces of muscles innervated by motor neuron Ds have more alpha-N-acetylgalactosamine and/or D-galactose than do those of muscles innervated by motor neuron Df. Biochemical analysis of the glycoprotein lectin receptors by sodium dodecylsulphate polyacrylamide gel electrophoresis has shown that approximately 10% of them are distributed among the various muscles in a manner that correlates with innervation by Df or Ds. Some of these macromolecules may be responsible for the biochemical differences in the muscle cell surfaces that could be specifically recognized by motor neurons. This intercellular recognition could mediate the reformation of the original innervation pattern during axonal regeneration.

Plasticity in the cockroach neuromuscular system

The retrograde transport of wheat germ agglutinin-conjugated horseradish peroxidase extracellularly injected into a leg muscle was used to identify the regenerating cockroach motor neurons that have grown an axonal branch into that muscle. At least 66% of the animals with crushed nerve roots eventually reform the original innervation pattern of this muscle with no mistakes. In spite of this apparent specificity the cockroach neuromuscular system can express plasticity as evidenced by the correction of mistakes made at early stages of regeneration. These mistakes are corrected through elimination during the time interval between 40 and 60 days after nerve crush. In addition, when the distal segments of the leg are removed, thus depriving some motor neurons of their normal target muscles, many of them form stable inappropriate axonal branches in denervated as well as fully innervated muscles. These observations are discussed in terms of possible mechanisms responsible for the specificity of the cellular interactions and in terms of their relevance to understanding the development of vertebrate neuromuscular systems.

Lectin receptors in the cockroach neuromuscular system. III. Distribution and identification within the thoracic ganglia of the nervous system

A fluorescence microscopic study of the binding of an array of 10 fluorescein isothiocyanate conjugated lectins to frozen sections of the cockroach thoracic ganglia was performed. Although a region of the neuropile receiving direct input from sensory neurons was observed to have distinctive lectin binding properties, no difference was detected between the lectin binding properties of Df and Ds, the two identified motor neurons that innervate the coxal depressor muscles in the leg. In addition, the three types of neurons identified in the ganglia, excitatory motor neurons, inhibitory motor neurons and dorsal unpaired median cells all had identical lectin binding properties. Therefore, in order to demonstrate the existence of macromolecules responsible for giving a biochemical identity to the various neurons it will be necessary to perform biochemical analyses of single cells or apply immunological techniques. A biochemical analysis of the ganglionic Con A and WGA receptors detected after fractionation by SDS polyacrylamide gel electrophoresis revealed the presence of some receptors not found in extracts of muscles and which may be specific for ganglia. In addition, a set of ganglionic lectin receptors were detected which were of similar solubility and lectin binding properties to a set of receptors found in muscle extracts and which disappeared from the muscles within one week of their denervation. It is suggested that such lectin receptors are found in all motor neurons and are transported to axon terminals within the muscles.

Thein VivoRegulation of Pioneer Axon Growth by FGF-2 and Heparan Sulfate Proteoglycans in Cultured Embryos of the Cockroach

Antibody perturbation experiments on cultured cockroach embryos demonstrated that a localized source of an FGF-2-like immunoreactive molecule in the head is required for the proper growth of pioneer axons in the leg. The study of axon growth in various fragments of cultured embryos and in the presence of various conditioned media showed that FGF-2 is needed to counteract the effects of an inhibitor of axon growth produced in the body trunk of the embryo. Endogenous heparan sulfate proteoglycans mediate these effects of FGF-2 on axon growth. The results of experiments with FGF-2 and/or body trunk axon growth inhibitor added to the culture medium indicate that more globally and uniformly distributed molecules may play as important a role in axon guidance as the more spatially restricted guidance cues. The results are interpreted in terms of a model that is consistent with a role for the FGF-2 receptor in axon growth.