Quantitative Staging of Embryonic Development of the Grasshopper, Schistocerca Nitens

J. Embryol. exp. Morph. Vol. 54, pp. 47-74, 1979 47 Printed in Great Britain © Company of Biologists Limited 1979 Quantitative staging of embryonic development of the grasshopper, Schistocerca nitens By DAVID BENTLEY,1 HAIG KESHISHIAN, MARTIN SHANKLAND AND ALMA TOROIAN-RAYMOND From the Department of Zoology, University of California, Berkeley SUMMARY 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 text), 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 %. INTRODUCTION Recently it has become possible to investigate the physiology, pharmacology, and morphology of single, identified neurons, neuroblasts and other cell types during embryogenesis in grasshoppers (Bate, 197'6 a, b; Spitzer, 1979; Goodman & Spitzer, 1979; Goodman, O'Shea, McCaman & Spitzer, 1979; Bentley & Toroian-Raymond, 1979). The paucity of preparations in which these approaches are feasible has made grasshopper embryogenesis particularly attractive for analysing many problems in developmental neurobiology and developmental biology in general. To accurately characterize the time course of developmental events, it is necessary to have a precise, rapid staging system, applicable to unstained, living material and covering the entire period of embryogenesis. Such a staging has not been available. There is an extensive literature on grasshopper embryogenesis extending from the mid-nineteenth century (Packard, 1883; Wheeler, 1893; Slifer, 1932a; Roonwal, 1936; Johannsen & Butt, 1941; Anderson, 1972). Several systems for staging development have been described (Table 3). Although various features of these systems are excellent, no single one covers the entire course of embryogenesis with the temporal and cellular detail now required. Most descriptions 1 Author's address: Department of Zoology, University of California, Berkeley, CA 94720, U.S.A. 48 D. BENTLEY AND OTHERS are illustrated by camera-lucida or free-hand drawings of whole eggs or embryos; many cover only a portion of embryogenesis. Most stages have been marked by easily observable changes in external morphology or orientation of the embryo; as these events are not distributed uniformly throughout development, relatively long, uncharacterized periods occur in these systems. In this paper, we present a new staging system. Its main features are: (1) the stages evenly subdivide the period of development; (2) stages are expressed as a percentage of total developmental time; (3) stages are illustrated by light photomicrographs; (4) stages are illustrated at three levels of increasing resolution (egg; embryo; limb). MATERIALS AND METHODS The experimental animals were Schistocerca nitens, initially captured in 1963 and maintained in culture for approximately 50 generations. The animals were raised in small, crowded cages at 31 ± 1 °C, 16L/8D light cycle, and 60 ± 5 % relative humidity on a diet of freshly sprouted wheat supplemented by wheat- germ and dry dog-food. Generation time was 10-12 weeks. Egg-pods were deposited in 6 cm diameter/10 cm high paper cups containing cleaned no. 1 sand moistened by 15 % water by weight. Cups were capped during incubation. Pods contained from 25 to 100 eggs. While pods used for maintaining the culture could be left undisturbed, those intended for experiments had to be opened. Two methods were used for accomplishing this. In the first, the pod was placed on top of the moist sand, broken into several clusters of exposed eggs, and covered with damp cotton; alternatively, the pod was com- pletely dispersed and eggs were individually washed in distilled water and placed separately on filter paper kept at a constant moisture level by capillary wetting. Eggs were kept at various temperatures in an incubator accurate to ± 0-5 °C, and at a relative humidity of 60 ± 2 %. We wanted to express developmental stage as a percentage of total developmental time, and further, to place the stages at equal intervals throughout embryogenesis. To accomplish this, it was necessary to have a group of syn- chronously developing eggs whose total developmental time could be accurately predicted. Sample eggs could then be withdrawn from this group at equal intervals (5 % of total developmental time in this case) and described. Eggs within the same pod formed our synchronous groups. The total developmental time of each egg was determined with an accuracy of ± 45 min. Grass- hopper eggs are fertilized when deposited (McNabb, 1928; Slifer & King, 1934) and completion of a pod takes about 1 h. We selected only pods where deposition was observed, so the time of fertilization could be determined to ± 30 min. Hatching time was established by continuously observing each pod and counting and removing all the nymphs which hatched within each 30 min period; the hatching time of each nymph was consequently known to ±15 min. This Quantitative staging of embryonic development of Schistocerca 49 information was obtained from ten pods, and formed the data for a quantitative characterization of the synchrony of hatching within pods (Figs. 1, 2). Five percent staging required the selection of 20 equally spaced observation times during the complete period of development. This was done by determining the temperature at which the eggs developed in 20 days, and then observing the eggs each day at the time of initial deposition of the pod. The appropriate temperature was predicted by observing the development of 192 pods at temperatures ranging from 30 to 35 °C. The actual developmental time of each of the pods used for staging was determined as described above. The staging descriptions are designed to be pre-experimental, and therefore are based on features which can be seen in unstained, living embryos with a dissecting microscope. Correspondingly, all photomicrographs were made with a dissecting microscope (Wild M5A; a few additional features which can be seen in simple squashes in a compound microscope are noted). A text description of each stage is provided as well as light micrographs at three levels of resolution: (1) the whole egg showing the size, location, and orientation of the embryo; (2) the embryo; (3) detail of the metathoracic leg. Eggs were immersed in 3 % sodium hypochlorite for 1 min; this procedure clears the chorion but doesn't remove it (longer exposure clears better but causes osmotic changes which alter the shape of the embryo). Cleared eggs were photographed in dark-field illumination; embryos were photographed in transmitted illumination or, after they became opaque, in incident illumination. All colors are described from incident illumination. The saline in which embryos were examined comprised NaCl 140 mM, KC1 5mM, CaCl2.2H2O 4mM, MgSO4.7H2O 2mM, TES 2 HIM, dextrose 55-65 mM, pH 7-2, osmolarity; 310-325 milliosmol/kg. High osmolarity was crucial to maintaining physiological condition and had to be adjusted for age of the embryo (Carlson, 1961; Grellet, 1968). It was altered by varying dextrose concentration to maximize heart rate, peristalsis rate, neuroblast mitosis frequency or, in early stages, to prevent shrinking or swelling of the amniotic cavity. The final staging is based upon precisely timed observation of eggs from eight pods. At each observation period, descriptions of several eggs from each pod were made; for three pods, photographs of several eggs were made each day. Confirmatory observations have been made on many additional pods. RESULTS The synchrony of hatching of eggs within the same pod is shown in Fig. 1. These four pods were maintained on sand under moist cotton (first method); the cotton was removed a few hours before the onset of hatching. The degree of synchrony can be expressed by calculating the percentage of eggs which hatched within a period equal to 1 % of total developmental time (Fig. 1). In three of four 50 D. BENTLEY AND OTHERS 10 Pod C Pod I) n = 50 7o= 100 '.'o= 100 •3 40 30 20 10 - 19 20 19 2G Developmental time (days) Fig. 1. Hatching synchrony of eggs within each of four pods (A, B, C, D; incubated on sand), n = the number of eggs hatching from each pod; % = the percentage of hatching eggs from each pod which hatched within a period equal to 1 % of the total development time. Quantitative staging of embryonic development of Schistocerca 51 20-5 Development time (days) Fig. 2. Hatching synchrony of eggs within each of four pods (E, F, G, H; incubated on filter paper), n = the number of eggs hatching from each pod; % = the percentage of hatching eggs from each pod which hatched within a period equal to 1 % of the total developmental time. In pod H, seven eggs hatched when all the eggs were wetted with cool water at day 19; all of the remaining eggs hatched within the 9 h period shown, but the exact time of hatching was not noted. pods, all the eggs hatched within this period. This degree of synchrony indicates that the time of hatching of a subset of eggs from a pod is an accurate estimate of when eggs removed for staging descriptions would have hatched if they had been left undisturbed. Therefore, the percentage of developmental time experienced by embryos withdrawn for staging can be estimated within an error ofl%. An additional problem is the degree to which synchronous hatching indicates synchronous development. Mechanisms, such as pheromonal or mechanical stimulation, might be present which initiate simultaneous hatching in eggs that are actually at slightly different stages of development.

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