Grasshopper Embryo Research Articles

Pathfinding by neuronal growth cones in grasshopper embryos. II. Selective fasciculation onto specific axonal pathways

In the previous paper (Raper, J.A., M. Bastiani, and C.S. Goodman (1983) J. Neurosci. 3:20-30) we showed that the growth cones of two sibling neurons, the G and C cells, follow the same route in the developing grasshopper neuropil until they reach a stereotypic choice point. Here their growth cones diverge from each other as G turns and extends anteriorly and C turns and extends posteriorly. In this paper we show that the G and C growth cones fasciculate and extend in opposite directions upon a specific bundle of four axons. This occurs even though many other axons are within filopodial reach of the G and C growth cones. We identified the four neurons (the A1, A2, P1, and P2 cells) whose axons form the bundle that G and C extend upon by filling individual axons with Lucifer Yellow and viewing the filled cells in living embryos and by filling individual neurons with HRP and reconstructing the axon bundle from electron micrographs. The G neuron extends anteriorly in the bundle containing these four axons; the C neuron extends posteriorly in the same bundle only after several other axons have joined in. These results suggest that the growth cones of the G and C neurons are determined to recognize and extend upon labeled axons, leading us to propose the "labeled pathways" hypothesis.

The neuroblast of the grasshopper embryo as a new mutagen test system: III. Chromosome breakage induced by cyclophosphamide is greater with activation by rat hepatocytes than by S12 mix

The promutagen cyclophosphamide (CPhos) induces chromosome breaks in neuroblasts of the 14-day-old grasshopper embryo ( Chortophaga viridifasciata DeGeer, Orthoptera: Acrididae) only when activated with S12 mix or freshly isolated hepatocytes. After 1-h exposure followed by 3-h recovery, CPhos + hepatocytes (from uninduced adult rats) induces about 5 times more acentric fragments and induces them at lower doses than does CPhos + S12 mix (from phenobarbital-induced rats). Both activation systems contained equivalent amounts of microsomal protein. Hepatocytes could be used in suspension immediately after isolation, thereby obviating the delay necessitated by allowing for attachment. In the absence of CPhos, with or without activators, no chromosome aberrations were observed. Without CPhos, hepatocytes are less toxic than is S12 mix, as determined by reduction in the number of dividing neuroblasts.

Guidance of neuronal growth cones: selective fasciculation in the grasshopper embryo.

Guidance of neuronal growth cones: selective fasciculation in the grasshopper embryo.

Pathfinding by neuronal growth cones in grasshopper embryos. I. Divergent choices made by the growth cones of sibling neurons

We are interested in how the growth cones of identified neurons navigate in the central nervous system of the grasshopper embryo. The behavior of identified growth cones was observed as a function of developmental time by (i) periodically removing embryos from synchronized clutches of eggs and (ii) filling identified neurons in the central nervous system with the fluorescent dye Lucifer Yellow. We examined the first six progeny of the identified neuronal precursor cell, neuroblast 7-4: the Q1, Q2, G, C, Q5, and Q6 neurons. Their growth cones extend sequentially upon the same route across the midline of the posterior commissure of the developing ganglionic neuropil. However, after reaching the contralateral side of the neuropil, their growth cones diverge from each other at reproducible, cell-specific choice points. By focusing our attention on one such choice point, at which the growth cone of the G neuron turns anteriorly while the growth cone of its sibling C neuron turns posteriorly, we hope to elucidate the environmental cues which guide individual growth cones.

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

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.

Afferent innervation shapes the dendritic branching pattern of the Medial Giant Interneuron in grasshopper embryos raised in culture

In the adult grasshopper the Medial Giant Interneuron (MGI) receives synaptic input from the peripheral sensory neurons of the cercus. We prevented this innervation in grasshopper embryos by cutting off one or both cerci at a stage when the first sensory axons are just beginning to reach the central nervous system (CNS), and the MGI has not yet formed its mature branching pattern. Following this operation the embryos were raised in vitro for 3–9 days, and the MGI injected with the fluorescent dye Lucifer Yellow to determine its morphology. The development of the deprived cells was then compared to that of the normal MGI (described in M. Shankland and C. S. Goodman, 1982, Develop. Biol., 92, 483–500) and of cultured, but unoperated, controls to ascertain whether these presynaptic axons influence the embryonic growth and branching of the MGI's dendrites. The results of these experiments show that dendrite formation is enhanced in regions of the neuropil containing sensory axon terminals and that the afferents exert their influence locally on restricted portions of the branching structure. The enhanced growth of innervated dendrites appears to occur at the expense of dendritic outgrowth elsewhere, suggesting that the growing dendrites may be competing for a limited supply of some cellular component necessary for continued growth. Thus, the MGI's final branching pattern is at least partially dictated by the spatial distribution of presynaptic axons within the embryonic nervous system.

Peripheral pathways are pioneered by an array of central and peripheral neurones in grasshopper embryos.

During embryonic development, neuronal growth cones traverse long distances along stereotyped pathways to arrive at their appropriate targets. Since Harrison's initial observations1, increasing evidence has supported the notion that axonal pathways are “pioneered” early in development when the distances are short and the terrain relatively simple; thus an axonal scaffold is erected upon which later neurones can navigate. Grasshopper embryos have emerged as an attractive preparation in which to study pathfinding because the nervous system is relatively simple, and the individual identified neurones in the periphery and central nervous system (CNS) of the embryo are large and highly accessible. How are the first axonal pathways established between the periphery and CNS? Bate2 has described a single pair of cells in the limb buds, and two pairs in the antennae, which were thought to establish the very first peripheral pathways over distances of several hundred micrometres from the periphery to CNS; he called these pairs of cells ‘pioneer neurones’. Keshishian3 confirmed this finding and also described4 a second pair of pioneers in the limb buds. We report here a further analysis of peripheral pathfinding in the grasshopper embryo using a monoclonal antibody, called I-5, which stains the cells responsible for establishing the first pathways5,6. In contrast to the earlier reports, we find a large array of peripheral cells responsible for establishing the first pathways, with both pathfinders and landmark cells spaced at short intervals along the route. Furthermore, growth cones from central motoneurones make important contributions to the first peripheral pathways6.

Antibodies to horseradish peroxidase as specific neuronal markers in Drosophila and in grasshopper embryos.

Antibodies specific for horseradish peroxidase (HRPeroxase) bind to neuronal membranes in Drosophila and serve as a specific neuronal marker. Immunocytochemical staining with these antibodies marks sensory neurons, peripheral nerves, and fiber tracks in the central nervous system of embryos, larvae, and adult flies. Similar patterns of staining also were seen in embryos of the grasshopper. It appears that an antigen associated with the nervous system and appearing early in differentiation is recognized by antibodies to HRPeroxase. Using this staining method, we followed embryogenesis of the central nervous system in Drosophila and found that the organization of central fiber tracks resembled that in the previously well-characterized grasshopper. We have used the anti-HRPeroxase antibodies to show that mutations affecting segmentation in Drosophila affect the organization of the embryonic nervous system.

The neuroblast of the grasshopper embryo as a new mutagen test system: I. In vitro radiosensitivity

The neuroblasts of the grasshopper embryo ( Chortophaga viridifasciata De Geer) are being studied to determine their suitability for detecting environmental clastogens (chromosome-breaking agents). They are very sensitive to the induction of chromosome breakage by radiation in vivo. Their sensitity, 0.011 break / cell / R, is 4–5 times higher than pollen mother cells of Tradescantia (micronuclei), 10 times higher than either human lymphocytes or Chinese hamster cells (metaphase chromosome aberrations), and 15 times higher than mouse than mouse erythroblasts (micronuclei). Furthermore, they have no spontaneous chromosome breakage, which facilitates the detection of agents that break chromosomes. The present study shows that Chortophaga embryos maintain normal mitotic activity in vitro for 5 cell cycles at 38°C (20 h), and that neuroblasts of embryos grown in vitro have the same radiosensitivity as those of embryos in vivo. Thus in vitro exposure of grasshopper embryos is a promising method for obtaining data on the response of neuroblasts to chemical clastogens.

Neuroblast of the grasshopper embryo as a new mutagen test system. II. Chromosome breakage induced by in vitro exposure of embryos to the direct-acting mutagens 4NQO, MNNG, adriamycin, and bleomycin.

The dose-response for the induction of acentric chromosome fragments was determined in neuroblasts of the grasshopper embryo (Chortophaga viridifasciata De Geer, Orthoptera: Acrididae) exposed in vitro to four direct-acting chemical known to be mutagenic, clastogenic, and carcinogenic: 4-nitroquinoline-1-oxide (4NQO), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), Adriamycin (ADM), and bleomycin (BLM). After a 1-hr exposure followed by a 3-hr recovery period (untreated cell cycle time is 4 hr), acentric fragments were observed at doses down to 1 microM 4NQO, 1.25 microM MNNG, and 0.125 microM ADM and BLM. After an 8-hr continuous exposure, acentric fragments were induced by 4NQO at a dose as low as 0.125 microM. These low concentrations also reduced the number of dividing cells. No chromosome aberrations or mitotic effects were observed in untreated embryos or in those exposed only to the solvent dimethyl sulfoxide. Because of the short cell cycle and the sensitivity of the neuroblast to the induction of acentric chromosome fragments by chemical clastogens, a minimum of time is needed to perform the test. From a comparison with the prominent clastogen test systems currently used, it is concluded that the grasshopper neuroblast test is the fastest and that it detects some agents that some systems do not. Grasshoppers have a worldwide distribution. If neuroblasts of other species prove to be as sensitive to mutagens as those of Chortophaga, investigators in many countries would have available a eukaryotic mutagen test system that is simple, fast, reproducible, and inexpensive.

Differentiation of an identified sensory neuron (SR) and associated structures (CTO) in grasshopper embryos.

The differentiation of an identified sensory neuron, the grasshopper wing hinge Stretch Receptor (SR), is examined throughout embryogenesis. The morphological features of the SR axon, as it finds its path from the peripheral cell body to the CNS, and the timing of this peripheral growth were determined by intracellular injection of Lucifer yellow. The course of growth of the SR axon within the CNS and the sequence of formation of the identified branches in its characteristic central arborization were investigated by silver intensification of cobalt-stained axons. In addition, intracellular recording from the cell body of SR was used to determine the onset of electrical excitability and the characteristics of the somal action potential. A brief account of the differentiation of the closely associated wing hinge Chordotonal Organ is also given. During differentiation, the cell bodies of SR and the first wing hinge Chordotonal Organ neuron (CTN1) extended processes and migrate posteriorly along a peripheral epithelial ridge. The cell bodies migrate up to 150 micrometers with a group of undifferentiated cells to a specific site at the posterior edge of the segment. As the SR and CTN1 cell bodies migrate, their trailing axons stretch across the epithelial ridge. These axons from the medial body wall nerve (1D2) that the axons of subsequent sensory neurons follow.

Development of a sensory afferent projection in the grasshopper embryo:II. Growth and branching of peripheral sensory axons within the central nervous system

The morphogenesis of several types of sensory axon branching patterns has been described by cobalt filling the cercal nerve of the grasshopper embryo at a series of different stages in development, thus staining the earliest sensory axons as they grow through the CNS. This embryonic sensory projection contains all five types of cercal afferents seen in the adult, and no new sensory tracts are added during postembryonic life. When the embryonic sensory axons first follow their pioneer axons into the neuropil they choose pathways which are characteristic of the adult sensory tracts. Since the afferents follow these paths without sending collaterals into the other tracts, it appears that the growth axon chooses its specific pathway without extensive exploration of alternative routes. Likewise, nearly all of the branches which arise from the embryonic sensory axons remain within the eventual domain characteristic of each cell type. This precise, determinate pattern of initial growth implies that the sensory axons are guided through the neuropil and achieve their final branching patterns with a minimum of overgrowth and pruning. The fact that initial growth is so precise also suggests that the parameters which guide the growing axon may help to determine its eventual pattern of synaptic connectivity by limiting its physical access to large portions of the neuropil which contain potentially compatible synaptic partner cells. Two different types of neurons may be supplying the sensory afferents with guidance cues: (i) Although most of the cercal sensory axons diverge from the cercal pioneer axons within the CNS, some sensory afferents continue to follow the pioneers through several ganglia. (ii) In the adult, a large number of the cercal sensory axons form a hollow shell of arborization around the main dendrite of an identified synaptic target cell, the Medical Giant Interneuron (MGI). This structure, the interneuron dendrite and the shell of sensory arbor, is called the cercal glomerulus. Since the MGI's dendrite is already present at the stage when the first sensory axons enter the CNS, interactions between these cells could serve to guide the glomerular sensory axons away from the pioneers into their future tracts.

Development of a sensory afferent projection in the grasshopper embryo:I. Growth of peripheral pioneer axons within the central nervous system

The grasshopper's cercal nerve is established early in embryogenesis by an identified pair of peripheral neurons called the cercal pioneers. Like the peripheral pioneer neurons in other insect appendages, these two cells send their axons from the periphery to the rudimentary CNS and thus lay the foundation for a nerve that will later be followed by a large number of sensory axons. In this paper, cobalt fills of the primordial cercal nerve were used to characterize the disposition of these peripheral pioneer axons within the embryonic CNS. The pioneer axons stained by this technique terminate in ellipsoidal growth cones which have filopodia radiating from the leading edge and a single long terminal filament pointing along the path the axon is taking. The growing axons also bear filopodia along their sides, but these structures disappear as the cells mature. The pioneer axons of the cercal nerve make an abrupt turn where they first enter the ganglion rudiment and join the axons of the primary longitudinal tract. The pioneers then grow along this tract for several hundred microns without forming secondary growth cones or branches. This prolonged absence of central arborization distinguishes the peripheral pioneer axons from the axons of later-arising epidermal sensory neurons.

The development of electrical properties of identified neurones in grasshopper embryos.

1. We have examined the development of the electrical properties of five identified neurones in grasshopper embryos between days 10 and 13 of embryogenesis (hatching occurs on day 20). DUM 3,4,5; DUM 4,5; DUM 5; the H cell; and the H cell sibling are the progeny of two different precursor cells. Electrical coupling and electrical excitability were assayed by intracellular recordings. 2. Midway through embryogenesis, on day 10, the five cells are highly electrically coupled to each other and are electrically inexcitable. The temporal sequence of the development of electrical excitability and electrical coupling is described for DUM 3,4,5; 4,5; and 5. The H cell and H cell sib undergo the same sequence one day later. 3. The first non-linear membrane property to appear is delayed rectification which appears on day 11 and can be blocked by tetraethylammonium (TEA). In some cells at about day 11, the addition of TEA to normal saline unmasks a Na+-dependent action potential in the axon. 4. The first action potential in normal saline is a Na+-dependent response that appears in the axon at day 11-11.5. 5. The next stage of excitability in normal saline is the appearance about day 11.5 of a Na+-dependent action potential in the median neurite between the soma and the two axons. In some cells at about day 11.5, the addition of TEA unmasks an excitable response in the soma. 6. Overshooting action potentials appear in the soma about day 12; the inward current is carried by both Na+ and Ca2+; TEA causes a prolonged shoulder on the falling phase of the action potential. A short time later, TEA causes a long-duration CA2+ plateau. 7. A progressive decrease in the degree of electrical coupling among the cells occurs between days 10 and 12.5. Complete uncoupling is never observed before day 11, but has always occurred by day 12.5. 8. Two methods were used to demonstrate that electrical coupling does not mask the presence of excitable inward current channels and thus make the cells appear inexcitable. First, we exposed the cells to veratridine. The cells which normally generate excitable Na+ response are depolarized by it; the younger inexcitable cells are not. Secondly, we electrically isolated the cells by killing the somata of their neighbours. The input resistance increased, yet the extent of excitability remained unchanged. 9. There is variability in the precise temporal relationship of excitability and uncoupling. Pairs of cells from different embryos of the same ate can generate the same type of action potentials and yet be coupled in one embryo and uncoupled in another. Electrical excitability and uncoupling appear to be causally unrelated and independent events, occurring at about the same developmental stage.

The mature electrical properties of identified neurones in grasshopper embryos.

1. We have examined the mature electrical properties of five identified neurones in embryos of the grasshopper Schistocerca nitens. The five cells arise from two different precursor cells: the median neuroblast, whose first three progency are called DUM 3,4,5; DUM 4,5; and DUM 5; and mid-line precursor 3, which divides once to produce the H cell and the H cell sibling.2. Electrical coupling was investigated by dual intracellular penetrations. Action potentials were elicited by intracellular stimulation of cell bodies and by extracellular stimulation of axons. The ionic basis of action potentials was investigated by changing the ionic environment and by applying various blocking agents.3. Most of the mature electrical properties of all five cells appear by day 13 of embryonic development. They change little through hatching on day 20.4. The recorded resting potential for all five cells varies from -55 to -60 mV and the recorded input resistance varies from 200 to 450 MOmega. All five cells show delayed rectification, much of which is blocked by tetraethylammonium (TEA). Their resistance increases as they are hyperpolarized.5. The five cells do not appear to be electrically coupled between days 13 and 20.6. All five neurones generate mature action potentials and in several cases show cell-specific electrical properties by day 13. The ionic dependence and depolarizing phase of the action potential change little between days 13 and 20; some changes occur in the after-hyperpolarization.7. One cell-specific difference is observed in normal saline. Four of the cells have axons, median neurites, and somata which generate action potentials in normal saline; but one of the cells (the H cell sibling) has an inexcitable soma and generates action potentials only in its axon.8. Another cell-specific difference in the soma action potentials of DUM 3,4,5,; DUM 4,5; DUM 5; and the H cell is observed when outward current is blocked by TEA. In three of the cells, TEA causes the short-duration action potential (2-4 msec) to be converted into a long-duration action potential (100-1000 msec) in which there is an initial spike (Na(+)-dependent) followed by a long plateau (Ca(2+)-dependent). In the other cell, DUM 5, at resting potential the addition of TEA only causes a shoulder (Ca(2+)-dependent) on the falling phase of the action potential. DUM 5 and DUM 4,5 thus have different electrical properties, even though they differ only by a single cell division from the precursor cell.9. In all four neurones which normally generate soma action potentials (DUM 3,4,5; DUM 4,5; DUM 5; and the H cell), the inward current is carried by both Na(+) and Ca(2+). On day 13, either inward current alone can generate the overshooting action potential; in contrast, by day 18, neither inward current alone can generate an overshooting response. The inward current of the axon action potential in all five cells is carried predominantly by Na(+).10. Thus, the progeny of two different embryonic precursor cells (median neuroblast and mid-line precursor 3) show a broad spectrum of electrical properties. The mature phenotype of electrical excitability is not a property shared in common by all the progeny of a single embryonic precursor cell in the grasshopper. Conversely, progeny from different precursor cells can share the same mature phenotype of electrical excitability.

The origin and morphogenesis of pioneer neurons in the grasshopper metathoracic leg

The origin and morphogenesis of pioneer neurons were examined in the developing metathoracic leg of cultured grasshopper embryos. The first two pioneer neurons to differentiate in the limb-bud were studied using continuous observation, time-lapse microphotography, and intracellular dye-fills. A single mother cell located within the ectodermal epithelium at the limb-bud tip divided symmetrically to yield the first pair of sister pioneer neurons. These neurons migrated out of the ectoderm in a morphologically undifferentiated state and initiated axonogenesis at a characteristic location between the ectoderm and the inner mesodermal epithelium of the limb-bud. Following a period of initially undirected filopodial sprouting, the axon pair grew proximally along a highly sterotyped path between the limb-bud ectoderm and mesoderm. The path taken by the pioneer axons did not follow any gross anatomical features of the adjacent limb-bud epithelia. The pioneer neurons described in this paper ultimately extend from the proximal end of the tibia into the metathoracic ganglion, and they probably give rise to nerve branch 5bl of the metathoracic leg.

Quantitative staging of embryonic development of the grasshopper, Schistocerca nitens

During development of the grasshopper embryo, it is feasible to examine the structure, pharmacology, and physiology of uniquely identified cells. These experiments require a fast, accurate staging system suitable for live embryos. We present a system comprising (1) subdivision of embryogenesis into equal periods, (2) expression of stage in percent of complete embryogenesis time, (3) characterization of stages by light micrographs (and descriptive test), and (4) illustration of stages at the egg, embryo, and limb levels of resolution. Advantages of a percent-system include communicability, flexibility in temporal resolution, accurate assignment of elapsed time in developmental processes, and uniform coverage of the period of embryogenesis. The stages described are at 5% intervals with an estimated error of +/- 1%.

Embryonic development of identified neurones: differentiation from neuroblast to neurone

Individually identified neurones are sufficiently large and accessible in grasshopper embryos to permit visualisation and impalement with intracellular microelectrodes from the time of their birth to their maturation. In this article part of the temporal pattern of differentiation from an identified neuroblast (precursor cell) to a group of identified neurones is described.

Embryonic development of identified neurons: temporal pattern of morphological and biochemical differentiation.

Individually identified neurons can be recognized in grasshopper embryos, and are accessible to examination by morphological, physiological, and biochemical techniques from their birth to their maturation. Only after the axon of an identified neuron reaches its postsynaptic target does the neurotransmitter accumulate, the soma rapidly enlarge, and the central arborizations greatly expand.

Effects of B chromosomes on development in grasshopper embryos

Pre- and post-diapause embryonic development was described in Myrmeleotettix maculatus in 24 stages up to hatching. The rate of development and mitotic index of 0B, 1B and 2B karyotypes in males and females were compared in the laboratory in material from two disjunct locations. The overall influence of one B chromosome on development does not appear to be major ; however, higher numbers of Bs have a more noticeable effect. The B chromosome quite clearly slows down the early prediapause development of male embryos, and individuals with 2B or 3B chromosomes are on average retarded in the later stages of the post-diapause period prior to hatching. The presence of B chromosomes produces a variety of chromosomally mutant embryos, many of which are arrested prior to diapause and few develop to hatching. The results are discussed in relation to the maintenance of the B chromosome polymorphism in this insect.