Ommatidium Assembly and Formation of the Retina-Lamina Projection in Interspecific Chimeras of Cockroach
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/. Embvycl. exp. Morph. Vol. 60, pp. 345-358, 1980 345 Printed in Great Britain © Company of Biologists Limited 1980 Ommatidium assembly and formation of the retina-lamina projection in interspecific chimeras of cockroach By MARK S. NOWEL1 From the Department of Zoology, University of Leicester SUMMARY By grafting operations, interspecific eye chimeras of the cockroaches Gromphadorhina portentosa andLeucophaea maderae were produced. Mechanisms involved in the development of both the compound eye and the retina-lamina projection have been studied. Most cell types composing the eyes of these cockroaches are cytologically distinguishable in the chimera; also, retinula axons forming the retina-lamina projection in the two species are of vastly different lengths. At the border between host and graft eye tissue, individual ommatidia are formed containing cells of both types. In particular, it is shov/n that the four cone cells can be found in any of the possible combinations of the two cell types. This shows that the cone cells within one ommatidium are not necessarily related by lineage. These results are in agreement with the hypothesis that cells within an ommatidium are determined by position rather than by a lineage mechanism. Furthermore, formation of mosaic ommatidia suggests that mechanisms governing eye formation are similar in these two species. The formation of the projection from donor retina to host lamina shows that axon elongation is not rigidly programmed, but that the axons grow until they reach a suitable target at which point connexions are made. INTRODUCTION By generating interspecific chimeras it is possible to investigate the question of whether pattern-forming mechanisms in different organisms are similar. In this study two species of cockroach, Gromphadorhina portentosa and Leucophaea maderae, have been used to create chimeric compound eyes. This experimental material enables us to investigate the specific question of cell lineage in develop- ment, because the eye cells in the two species are cytologically identifiable. It also enables us to ask the general question of whether or not ommatidia in different genera are formed by the same mechanisms. Finally, the compound eye as a component of the nervous system has enabled us to examine questions concerning nerve growth in a chimera. Concerning the question of cell lineage, it has been possible to confirm the observations of previous workers that there is no causal relationship between lineage and determination in the formation of ommatidia (Yagi & Koyama, 1 Author's address: Department of Zoology, University of Leicester, Leicester LEI 7RH, U.K. 346 M. S. NOWEL 1963; Hanson, Ready & Benzer, 1972; Ready, 1973; Benzer, 1973; Shelton & Lawrence, 1974; Green & Lawrence, 1975; Ready, Hanson & Benzer, 1976; Nardi, 1977; Shelton, Anderson & Eley, 1977; Lawrence & Green, 1979). In particular, cone-cell lineages have been examined. At the borders of graft and host tissues, ommatidia are found containing cells of both genotypes. The two types of cells behave autonomously and mosaic ommatidia have normal numbers of cells even though some are of one genotype and some are of the other. Thus, whichever pattern-forming mechanism is responsible for ommati- dium assembly, it is the same in these different genera. Concerning nerve growth, the mode of establishment of connexions between the eye and optic lobe has been examined. In the two species, retinula axons growing into the optic lobe have to grow different distances to form their connexions. In an interspecific chimera, L. maderae retinal tissue is three times farther away from its target, the lamina of its G. portentosa host, than it would be from its own lamina in the unoperated situation. Nevertheless, retinula axons from the foreign retina can form connexions in the lamina of the under- lying optic lobe. This provides valuable information on factors governing nerve growth and target discrimination. MATERIALS AND METHODS Cultures of G. portentosa and L. maderae were maintained under conditions of constant temperature (24 °C) and an alternating cycle of 12 h light/12 h dark, and fed on a diet of rat pellets and water. Grafts of eye margin and adjacent vertex epidermis were exchanged between young, newly moulted G. portentosa and L. maderae nymphs. The animals were anaesthetized by cooling in ice for 10-20 min and restrained with strips of Plasticine on a bed of moulded Plasticine. Operations were carried out under a dissecting microscope. Excisions of the integument were made using a razor blade fragment (Gillette francais) supported in a pin vice, and grafts were transferred from donor to host site using tungsten needles. After positioning the graft in the host site which had been kept moist with insect saline (Hoyle, 1953), a small droplet of insect wax (Krogh & Weis-Fogh, 1951) was used to seal the operation site. Operated animals were allowed to grow to the imago. To map areas of neural projection from graft-derived ommatidia in the chimeric eye into the lamina neuropile of the optic lobe, small localized lesions were made within the graft-derived eye tissue of the adults. After immobilizing the anaesthetized cockroach as for a grafting operation, a silver earthing wire was inserted into the head through a hole cut in the cuticle at a posterior medial point. A tungsten microelectrode was connected to a function generator set to deliver 2 /iA at a frequency of 1 MHz. After removal of narrow strips of the overlying cornea, the microelectrode was inserted into the exposed ommatidia to be electrolytically destroyed, and left in each point of insertion for 10 sec. The Cockroach compound-eye development 347 wounds were covered with insect wax. Sixteen hours later the animal was killed and the eye and optic lobe were fixed in a mixture of glutaraldehyde and paraformaldehyde (Karnovsky, 1965) in a 0-1 M-phosphate buffer (Hayat, 1970) at pH 7-4 for 2-4 h. The tissue was post-fixed in phosphate-buffered 1 % osmium tetroxide for 2-12 h, then dehydrated in an acetone series, cleared in propylene oxide, and embedded in Spurr's resin following a long period of infiltration. The optic lobe was serially sectioned in 1 ^m horizontal sections with a Huxley Ultramicrotome and glass knives, mounted in order on subbed slides, stained with toluidine blue, and examined with a Zeiss compound microscope for degenerating retinula axon terminals which appear dark blue following such treatment (Geisert & Altner, 1974). Chimeric eyes were sectioned perpendicular to the ommatidial long axis at the graft/host border. Serial semithin (1 ju,m) sections were collected in order on subbed slides and stained with toluidine blue. Ultrathin (80-100 nm) sections cut on a Dupont diamond knife were collected on collodion films (Pease, 1964), placed on to slot grids, stained with uranyl acetate and Reynolds' (1963) lead citrate, and examined with an AE1-802 Electron Microscope or a Siemens 102 Elmiskop. For wax histology, quarter heads were fixed for at least 3 h in alcoholic Bouin's fluid (Dubosq-Brasil) (Pantin, 1969) and left in 70 % isopropyl alcohol for periods of several days to several weeks to wash out the fixative and to soften the cuticle. After dehydration and embedding in paraffin wax, 10 /*m thick horizontal sections were cut on a Cambridge rocking microtome, dried on to albuminized slides and stained using a modification of the Mallory- Azan technique (Schiimperli, 1977). Experimental animals were photographed using a Zeiss Tessovar Photo- macrographic Zoom system. Sectioned material was photographed on a Zeiss Photomicroscope II. RESULTS Identification of cell genotypes in G. portentosa and L. maderae Sections through ommatidia in adult G. portentosa and L. maderae at the level of the crystalline cones are shown in Figs. 1 and 2. Cone cells in G. porten- tosa have a denser granularity and are considerably larger than those of L. maderae: crystalline cones (composed of four Semper's or cone cells) in G. portentosa measure approximately 35-40 pm in diameter at their bases with a base to apex length of 60 [im, while those of L. maderae measure 25 /im in diameter with a base to apex length of 35-40 /*m. In the chimeric situation (Figs. 5-11) the relative sizes of not only the cone cells (Figs. 5-8) but also the pigment granules which provide distinguishing features between both the retinula and primary pigment cells of the two species (Figs. 7, 10) are easily seen. In the primary pigment cells, pigment grains of G. portentosa are large 348 M. S. NOWEL FIGURES 1 AND 2 Semithin sections through the compound eyes of G. portentosa (Fig. 1) and L. maderae (Fig. 2) cut perpendicular to the ommatidial long axis at the level of the crystalline cones (cc). The Semper's cells comprising the crystalline cones may be distinguished on the basis of their relative sizes: cones of G. portentosa are com- posed of four large cells, while those of L. maderae are made up of four smaller cells. Each crystalline cone is surrounded by two primary pigment cells (pp) and numerous secondary pigment cells (sp). (The basis of the differential staining of cone cells within the same cone, often observed, is not understood.) Bars represent 10/*m. Cockroach compound-eye development 349 (1-3-1-7/mi) while those of L. maderae are slightly smaller (1-0-1-2/mi). Pigmentation of the retinula cells is similarly distinguishable: larger (0-7- 1-0 /mi) pigment grains in G. portentosa cells, smaller (0-35-0-5 /mi) grains in those of L. maderae. Secondary pigment cells are not easily distinguishable. Interspecific mosaic ommatidia Of approximately 400 grafting operations, approximately 20 resulted in interspecific chimeras. Following such operations, L. maderae eye tissue appears to grow at a slightly faster rate and G. portentosa at a slightly slower rate than does their host eye tissue.