DEVELOPMENTAL
BIOLOGY
71,142-152 (19%)
Cell Lineage in the Developing
Retina of Drosophila
A. LAWRENCE AND SHEILA M. GREEN of Molecular Biology, Hills Road, Cambridge, CB2 2QH, England
PETER MRC Laboratory
Received December 15, 1978; accepted January 31, 1979 Analysis of the cell lineage of the Drosophila retina is reported. Mitotic recombination within the white locus results in the formation of small red spots in white eyes; these are found under the dissecting microscope. The spot frequency is low (never more than l/30 eyes) so that there can be no doubt that each spot is a single clone. Eyes bearing a clone are serially sectioned and all retinula and all pigment cells scored as white or white’. We describe the constitution of 101 clones and examine the disposition of the marked cells in the retinal lattice. The clones are apparently random combinations of the marked cell types-for example, two-celled clones containing one pigment and one retinula cell are frequently found. Our results appear to rule out fixed cell lineage as a determinative mechanism in ommatidial development. INTRODUCTION
The intricate array of cells in the insect retina is so precise that it has been compared to a crystal (Benzer, 1973). In Drosophila the unit cell of this “crystal” consists of eight photoreceptive retinula cells, four cone cells, a four-celled bristle (Waddington and Perry, 1960), two primary pigment cells, and about four other pigment cells (Ready et al., 1976). The precision of this lattice led to the idea that it would develop by a precise cell lineage (Bernard, 1937; Kuhn, 1965). From studies with mosaic eyes, we now know that in Drosophila (Hanson et al., 1972), Oncopeltus (Shelton and Lawrence, 1974), and Periplaneta (Shelton et al., 1977) this mechanism does not operate: The cell lineages of ommatidia are indeterminate, each ommatidium descending from several precursor cells. These studies led to the conclusion that “cell determination within ommatidia is not connected with cell lineage but is dependent on cell position within the developing ommatidium” (Shelton and Lawrence, 1974). In Drosophila and the locust (Anderson, 1978) the formation of ommatidia is a gradual process, with certain cell types being generated before others-for example, in Drosophila, retinula cells 1, 6, and 7 142 0012-1606/79/070142-11$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
are added to preexisting clusters of cells at the end of eye development (Ready, 1973; Ready et al., 1976; Campos-Ortega and Gateff, 1976) so that in the development of the array “the final cell type may be determined according to the lattice position into which the cell is recruited” (Ready et a& 1976). It has been pointed out by Campos-Ortega that these studies do not exclude some lineage rules, which might operate in the last few cell divisions. For example, the developing retina could be subdivided into two sets of cells, one generating only pigment and one generating only retinula elements-the progeny of these cells would then assort themselves appropriately as the ommatidial clusters form (Campos-Ortega and Hofbauer, 1977). Lineage rules of this kind should be detectable in clones made during the last few cell divisions; one such approach has been employed (Campos-Ortega and Hofbauer, 1977; Campos-Ortega et al., 1978) but is subject to technical difficulties. Nevertheless, the authors concluded that their results “strongly suggest that fixed cell lineages are involved in the development of ommatidia.” Here we describe a new approach which obviates most of the technical problems.
LAWRENCE AND GREEN
Development of Ommatidia
143
optics. phase-contrast and bright-field Some clones were also examined in the electron microscope. Not all clones were successfully sectioned, the most frequent MATERIALS AND METHODS cause of failure being poor fixation-some We have used the method of Stern (1969) clones were lost because of this, especially of producing white+ clones in white eyes, during the early phase of our work. Screenby generating mitotic recombination within ing was done independently by the two the white locus. Two different white alleles authors-there was almost complete agreeare arranged in trans, such that mitotic ment (>90% of clones) when notes were recombination between them can form a compared. The few cases of disagreement wild-type chromosome (Fig. 1). w’ 0 were were resolved or, in two cases, discarded crossed to w65a256 and eggs collected and because we felt the preservation was inadirradiations of 1000 R performed in the equate. In many cases we were unable to usual way (Lawrence and Morata, 1977). distinguish between secondary and tertiary The time of irradiation was fixed in number pigment cells (Ready et al., 1976); in the of hours before puparium formation (hr tables we treat them as one cell type. BPF) (Garcia-Bellido and Merriam, 1971). Fl 0 were screened under the dissecting Assessment of Screening Efficiency microscope and clones containing one or The sections were examined for marked more pigment cells could be seen (but not pigment or retinula cells; all eight retinula those containing only retinula cells). The Rl-R8 (Dietrich, 1909) and all pigment first observer always missed some clones cells could be scored (Fig. 3 shows arrangeand therefore all flies were screened at least ment of the cells). When there were blocks twice and usually three times. The third of marked secondary and tertiary pigment observer still found a few clones so some cells, there was occasionally a problem in clones must have been missed. In one ex- determining the exact number markedperimental series 500 unirradiated controls deeper sections were helpful here, as the were included without the observers know- cells could be individually scored as discrete ing which flies had been irradiated-3 clusters of granules. The best region for clones were found in 1000 control eyes.’ scoring retinula cells R8 was at their most Eyes containing clones were fixed in half- distal extent and it was also helpful that strength Karnovsky (1965) fixative (see these cells had larger pigment granules than Ready et al., 1976) and embedded in aral- the other retinula cells (Rl-R7). After dite. The blocks were oriented so that the study of the selected clone, the remainder clone would be cut at right angles to the of the eye was carefully screened for other long axes of the ommatidia and serially clones. The fraction of each eye screened sectioned at 2 pm for observation using (usually 2/) was recorded, so that the sum ’ Stern’s purpose was to estimate the spontaneous total effectively searched for additional frequency of mitotic recombination. He found only 4 clones could be used below. In one series clones in 6137 flies, whereas our yield (3/500) is some (irradiation at 28 f 4 hr BPF) where the 10 times higher-the cause of the difference probably clone frequency detected under the dissectbeing the diffkulty of detecting very small clones. ing microscope was 1 clone per 30 eyes, 53 Counterbalancing this was the assumption (made by Stern in his calculations) that ommatidia had to be eyes were successfully sectioned and a entirely pigmented to be storable. There are six pigsummed area equivalent to 34 eyes was ment cells per ommatidium which if singly labelled screened. One eye had a second clone which can be detected, so that his calculated frequency of included a marked pigment cell, and two mitotic recombination (0.5-l/10”) was perhaps only a eyes contained additional clones of retinula slight underestimate.
Our results appear to rule out rigid cell lineages as a determinative mechanism in the development of ommatidial cell types.
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DEVELOPMENTAL BIOLOGY
cells only. Of the other 119 eyes that were sectioned (irradiated at diverse ages >56 hr BPF), no clones that had been overlooked down the dissecting microscope were found, with the sole exception of one clone containing three retinula cells (irradiation at 39 + 1 hr BPF). We can conclude that (i) the screening for clones is efficient when they contain pigment cells and (ii) clones that were exclusively retinula cells were not detected down the dissecting microscope. RESULTS
VOLUME 71,1979
frequency and size should be reciprocally related, so that one would have expected the frequency at 28 + 4 hr BPF to be more than twice that at 52 + 4 hr BPF. In fact, it is only some 50% higher-probably because a much larger fraction of the clones produced at the later time consists of retinula cells only, and is therefore missed in the screening. The lower frequency and small size of clones found after irradiation at 4 f 4 hr BPF suggest that cell division in the eye has nearly ceased by that time. Position of Clones
Frequency and Size of Clones After pilot studies, certain ages of irradiation were chosen for detailed analysis, and the mean frequency and size of clones produced at these times are shown in Table 1. The log size of clones decreases as pupation approaches, until at 4 f 4 hr BPF most of the clones found are of one cell only. Clone
Direct observation (Becker, 1957; Ready et al., 1976) has shown that the eye develops in an orderly manner from posterior to anterior. By the middle of the third instar, ommatidial clusters can be detected at the posterior margin of the eye, and progressively, clusters appear more anteriorly. The process is completed shortly after pupation. w65a25 white
,65a25
we
red
FIG. 1. Mitotic
recombination
within
the white locus can generate a white’ TABLE
chromosome.
1
VARIATION OF CLONE FREQUENCY AND SIZE WITH AGE AT IRRADIATION No. of Age (hr BPF) No. of eyes” Frequency* Log size f SE clones” Unirradiated controls 3 loo0 0.003 4*4 19 1406 0.013 0.06 + 0.03 28 f4 89 2614 0.034 0.32 + 0.04 2736 0.022 0.73 + 0.07 59 52 + 4
Mean sizeb 1.2' 2.1 5.3
’ These numbers are based on clones found under the dissecting microscope, not all of which were successfully sectioned. b Size estimated from eyes successfully sectioned (Tables 2 and 3). 'N=14.
Development
LAWRENCE AND GREEN
ula cell precursors (Campos-Ortega and Hofbauer, 1977), small clones should contain either retinula cells or pigment cells, but not both. Table 2 gives the constitution of the clones found at this time, and it would appear that clones can be of many combinations, although because of the screening method, all contained at least one pigment cell. For example, clones 113, 123, 204,205, and 210 are of only two cells each, one pigment cell and one retinula cell. One might expect segregation between the polar retinula cells (R2-5) and the equatorial ones (Rl, 6, and 7), but this is not supported by the data (e.g., clones 120 and 230). Most small clones contained only one example of each retinula cell type, but two small clones showed that this is not a rule. One (made at 33 + 8 hr BPF) was of four cells, one primary and one secondary pigment cell and two retinula cells Rl, while another (195, made at 52 + 4 hr BPF) was of only four cells yet had two retinula cells R2 (Fig. 4e). Those clones produced at 52 f 4 hr BPF are on average larger (Table 3) and therefore not so ideal for revealing late lineage restrictions, but they are consistent with the earlier clones. They show no pattern and sometimes include several examples of the same cell type, even when other cell types are not represented (e.g., 196).
In all aspects the posterior part of the eye develops in advance of the more anterior parts, with waves of mitoses moving across the eye from posterior to anterior (Ready et al., 1976). Accordingly, one would expect clones induced at any one time during this process to vary in size depending on their position within the eye, anterior clones being larger; the positions and sizes of the clones were therefore plotted as accurately as possible on standard eye diagrams (Fig. 2). At 52 & 4 and 28 f 4 hr BPF the clones are distributed over most of the eye, and there is an approximate relationship between clone size and position. At 28 f 4 hr BPF the four largest clones (9, 10, 11, and 13) are found near the anterior margin, with the smaller clones being more common posteriorly. The most posterior part of the eye is free of clones. At 4 +- 4 hr BPF the few clones are mostly confined to the anterior part of the eye. Constitution of Clones Examples of the preparations are shown in Figs. 4 and 5. Many of the clones produced at 28 + 4 hr BPF and at later times were small, the largest being 13 cells. It is here that one might expect lineage restrictions to show; for example, if there were a segregation between pigment cell and retin424hBF’F
D
v
145
of Ommatidia
28 t4hPPF
52+4hBF’F
D
D
v
v
FIG. 2. Distribution on eye of clones produced at three ages. The clones were originally plotted, by deadreckoning on a basis of ommatidial row coordinates, on a standard eye diagram. Each number represents the position of one clone containing that number of cells. A, anterior; P, posterior; D, dorsal; V, ventral.
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TABLE 2 CLONES PRODUCED BY IRRADIATION AT 28 f 4 HR BPF”
Clone No. 376 4ob
109 113 122 123 124 173 178 200 204 205 207 208 210 237 238 251 43b 107 114 175 231 65 106 203 230 73 176 201 71 172 206 120 177 74 234 250
Number cells 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 4 4 4 4 5 5 5 6 6 6 7
9 10 11 13 150
I
II + III
Rl
R6
R7
R2
R3
R4
R5
R.8
2 1 1 1
1 1 1 1 1
1
1 1 1 1
1 2 1 1 1 2 1 2 1 2 27
I 1 1 2 1 1 1 2 1 1
1
1 1
1 2 1 1 1 1 3 3 1 1 1 2 3 1 5 2 2 3 4 3 2 3 2 5 5 70
1 1 1
1 1
1
1 1
NS’
1 1 1 1 1
1 1 1
1
1
1
1 1 1 1
I
1 1 1 1 9
1 8
1 1
4
1 1 2 6
1 1 3
1 1 1 1 1 11
1 1
NS
1 8
1 4
DIn the same series 23 single-cell clones were found; of these, 8 were primary pigment cells (I) and 14 were secondary (II) or tertiary (III). There was one clone of 1 and one clone of 2 retinula cells in these eyes. ’ Irradiated 20 f 4 hr BPF. ’ Not storable.
It is known (Ready et al., 1976; CamposOrtega and Hofbauer, 1977) that only the pigment cells and retinula cells Rl, R6, and R7 are formed by the final mitotic front to sweep across the eye from posterior to anterior. The other retinula cells are formed in previous mitoses. One would therefore expect that clones would include labelled
Rl, R6, and R7 cells more often (CamposOrtega and Hofbauer, 1977). However, this is only true if a sufficient number of the clones is produced in cells in a stage after the formation of the R2-R5 and R8 cells but before or during the generation of the Rl, R6, and R7 cells. As only a small proportion of each irradiated eye is actually in
LAWRENCE AND GBEEN
147
Development of Ommatidia
TABLE
3
CLONES PRODUCED BY IRRADIATION AT 52 f 4 HR BPF Clone No. 131 220B 243 247 69 216 222 223 242 244 130B 184 185 188 195 212 224 198’ 246 197 215 183 171 217 130A 182 245 241 189 214 196 220A 186 190
-
Number cells 2 2 2 2 3 3 3 3 3 3 4 4 4 4 4 4 4 6 6 8 8 9 10 10 11 11 14 16 19 19 23 29 32 55 340
II + III
I
Rl
R7
R6
R2
R3
___~ R4
R5
R8
2 1
1
1 1 1 1 1 2 2 2
1 1
1 2 1 1 1 2 1 3 1 2 2 2 3 1 1 2 3 2 3 5 4 9 61
1 2 1 1 5 2 1 2 2 2 5 3 7 8 5 9 7 12 7 10 15 122
1
1 1 1
1 1
1 1 1 2
1
20
-
1
1
1
2
1 1 1 2 1 2 2 4 18
1 1
1 2 1 1 1 3 2 16
1
1
1 1
1
1 1 1 2 2 1 1 2 2 3 5
1 1 2
2 1 3 1 5 17
a In the same series four single-cell clones of primary (I), secondary (II), or tertiary (III) pigment cells were found; there were no other clones. Two pairs of clones (130A, 130B; 220A, 220B) were each found in one eye. * On extreme edge of eye.
this stage, one might not expect the preferential labelling of Rl, R6, and R7 to be detectable (Tables 2 and 3). The border of large clones, or patches in gynandromorphs (Benzer, 1973; Ready et al., 1976), is very uneven, showing mosaicism spread over about two ommatidial rows. It is therefore no surprise that the small clones are made up of somewhat scattered cells. Usually the clones are confined to a patch consisting of a central plus the six adjacent ommatidia, but within this area they can be well separated. Some examples
are shown in Fig. 3, while a series of sections of one clone (183) is illustrated in Figs. 4ad. We have found five clones at the extreme edge of the eye. These were not included in the tables, as they, with one exception (Fig. 4f), contained only peripheral pigment cells. They confirm (Ready et al., 1976) that the peripheral ommatidia are surrounded by a continuous sheath of pigment cells. Roughening X irradiation
produces a band of rough-
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DEVELOPMENTAL BIOLOGY
LEFT EYE DORSAL
VOLUME 71,1979
RIGHT EYE DORSAL
FIG. 3. Examples of 10 clones, to show the distribution of marked cells (shown in black) in the lattice. The position of the clone in the eye (right R or left L, dorsal D or ventral V) is given as well as the clone number (see Tables 2 and 3). Orientation sketches to show numbering and nomenclature (from Ready et al., 1976) are given at the top. 1-8, Retinula cells; HNG, hair nerve group; I, II, and III, primary, secondary, and tertiary pigment cells; A, anterior; P, posterior. Clones 186 and 123 are at the edge of the eye.
FIG. 4. (a-d) Sections of clone 183 (compare Fig. 31 at four different levels. (a) Level of the primary pigme bnt cells; the two marked primaries (I), a secondary (II), and a tertiary (III) are seen. (b) At the most distal part Of the retinula cells, one marked retinula cell R4 is arrowed. (c) Lower in the retinula cells; solid arrows point to marked retinula cells, and open arrows to unmarked ones. In white’ retinula cells the pigment granules Ewe found clustered at the base of the rhabdomere and scattered elsewhere in the cell. (d) At the level of the 1K8 cells, one is labelled. The marked secondary and tertiary pigment cells can be traced through all four sectioi 1s. (e) Clone 195 (Table 3) showing two labelled retinula cells R2 and one H3. (0 Section through clone 198 (Tat de 3) at extreme edge of eye; this clone marks five secondary pigment cells and one primary pigment cell (not in plane of section). Sections ca. 2 pm; phase contrast. x 1200. 149
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FIG. 5. Electron micrograph of section through clone 65 (Table 2). Large pigment granules are present in the marked secondary pigment cell (II), and smaller ones (p) in the marked retinula cells R4 and R5. Large numbers indicate the rhabdomeres, and smaller ones the cytoplasm of the retinula cells RI-R7. Arrows indicate attachment desmosomes between neighbouring retinula cells.
LAWRENCE AND GREEN
ened ommatidia parallel to the dorsoventral axis, its position moving anteriorly with later irradiations (Becker, 1957). This roughening is associated with defective ommatidia which lack one or more cells (Campos-Ortega and Hofbauer, 1977). In our hands, the roughening was variable. In one case only, marked cells were found in a defective ommatidium, but in other cases roughened ommatidia were found near to the clone (one is shown in Fig. 3, 205). If rough ommatidia were plotted in relation to the clone and different eyes compared, it was found that there was no simple spatial relationship. For example, the band of roughening, which could affect as many as 20 ommatidia in some eyes, was usually posterior to the clone, but rough ommatidia were also found near the clone and anterior to it. DISCUSSION
Small red spots are generated in the developing white retina of Drosophila, and their size and constitution recorded. Because the spots are rare (never more than 1 spot/30 eyes), each spot must be an individual clone. Even when produced at the very end of retinal development, the clones show no evidence for rigid cell lineages, and label the pigment and retinula cells in diverse combinations. These results support earlier views of ommatidia development (Ready et al., 1976; Shelton and Lawrence, 1974) which envisage local cell migration and an orderly assembly of uncommitted cells into the lattice. Rigid cell lineages in the development of ommatidia have been proposed by ComposOrtega and colleagues after an extensive series of experiments (Hofbauer and Campos-Ortega, 1976; Campos-Ortega and Hofbauer, 1977; Campos-Ortega et al., 1978). These authors made clones of recessive markers such as white and rdgB in heterozygous eyes of wild-type phenotype. One problem was that with white clones there was sometimes difficulty in scoring the lack of pigment in the retinula cells (CamposOrtega and Hofbauer, 1977). Also, because
Development
of
Ommatidia
151
of the large number of spots found per eye, an operational criterion had to be devised to define when a spot should be treated as a clone; this was done when marked cells were confined to a group of seven ommatidia (a central one and its six immediate neighbours). This operational criterion is suspect: For example, Fig. 3 shows one instance (197) which would have been classified as two clones (Campos-Ortega and Hofbauer did not score primary pigment cells). Further, evidence that some of the spots which do meet the criterion are nevertheless two clones is presented by CamposOrtega et al. (1978). These authors conclude that double hits could explain all cases of “clones” containing both retinula and pigment cells. However, we have several cases where small clones do contain both types of cell (Tables 2 and 3) and no suggestion that there is any segregation. The clearest examples are those clones of only two cells, one retinula and one pigment cell (40,113,123,204,205,210,237, and 238, Table 2; 220B, 243, and 247, Table 3). Their operational criterion may also have led these authors to occasionally classify two clones as one, and these problems may well be the root cause of the many discrepancies between their results and ours. The assessment of cell lineage by clonal analysis requires certain discrimination between single clones and double events; without this discrimination, conclusions can be insecure. We may not have enough clones to test all possible lineage rules, but Tables 2 and 3 list sufficient to exclude all simple ones. Our results would appear to rule out cell lineage as a determinative mechanism in the development of ommatidia. This conclusion refers only to those cell types we were able to score (the hair nerve groups and cone cells were not scored). Ommatidial assembly is probably normally associated with some cell death (Spreij, 1971), and no doubt the X irradiation kills many additional cells. Nevertheless, apart from a few roughened ommatidia, the pattern is perfect-an observation which supports our view that
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DIETRICH, W. (1909). Die Facettenaugen der Dipteran. Z. Wiss. Zool. 92,465-539. GARCIA-BELLIDO,A., and MERRIAM, J. (1971). Parameters of the wing imaginal disc development of Drosophila melanogaster. Develop. Biol. 24, 61-87. HANSON,T. E., READY, D. F., and BENZER, S. (1972). Use of mosaics in the analysis of pattern formation in the retina of Drosophila. Caltech Biol. Annu. Rep. 40. HOFBAUER, A., and CAMPOS-ORTEGA,J. A. (1976). Cell clones and pattern formation: genetic eye mosaics in Drosophila melanogaster. Wilhelm Roux Arch. 179, 275-289. KARNOVSKY, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27, 137A-138A. KIMBLE, J. E., and HIRSH, D. (1979). The postembryonic cell lineages of the hermaphrodite and male gonads in C. eleguns. Develop. Biol. (in press). KUHN, A. (1965). “Vorlesungen iiber Entwicklungsphysiologie.” Springer-Verlag, Berlin. LAWRENCE,P. A. (1966). Development and determination of hairs and bristles in the milkweed bug Oncopeltus fasciutus, Dall. J. Exp. Biol. 44, 507We thank Jose Campos-Ortega for advice on tech522. niques and discussion; Jonathon Hodgkin, Judith Kimble, and John Sulston for help with the manu- LAWRENCE, P. A., and MORATA, G. (1977). The early development of mesothoracic compartments in Droscript; Gary Struhl and John White for encouragesophila. An analysis of cell lineage and fate mapping ment; and Paul Johnston for assistance with the and an assessment of methods. Develop. Biol. 56, screening. 40-51. REFERENCES LEES, A. D., and WADDINGTON, C. H. (1942). The ANDERSON,H. (1978). Postembryonic development of development of bristles in normal and some mutant the visual system of the locust, Schistocerca gretypes of Drosophila melanoguster. Proc. Roy. Sot. garia. I. Patterns of growth and developmental London B 131,87-110. interactions in the retina and optic lobe. J. Embtyol. READY, D. F. (1973). Pattern formation of the retina Exp. Morphol. 45, 55-83. in Drosophila. Caltech Biol. Annu. Rep. 203. BECKER, H. J. (1957). Uber Rontgenmosaikflecken READY, D. F., HANSON, T. E., and BENZER, S. (1976). und Defektmutationen am Auge von Drosophila Development of the Drosophila retina, a neurocrysund die Entwicklungsphysiologie des Auges. Z. Intalline lattice. Develop. Biol. 53, 217-240. dukt Abst. Vererb. 88, 333-373. SHELTON, P. M. J., ANDERSON, H. J., and ELEY, S. BENZER, S. (1973). Genetic dissection of behavior. Sci. (1977). Cell lineage and cell determination in the Amer. 229,24-37. developing compound eye of the cockroach, PeriBERNARD, F. (1937). Recherches sur la mophogenie plunetu americana. J. Embryo1 Exp. Morphol. 39, des yeux composes d’arthropodes. Bull. Biol. Fr. 235-252. Belg. Suppl. 23. SHELTON, P. M. J., and LAWRENCE, P. A. (1974). CAMPOS-ORTEGA,J. A., and GATEFF, E. (1976). The Structure and development of ommatidia in Oncodevelopment of ommatidial patterning in metamorpeltus fasciutus. J. Embryol. Exp. Morphol. 32, phosed eye imaginal disc implants of Drosophila 337-353. melanogaster. Wilhelm Roux Arch. 179, 373-392. CAMPOS-ORTEGA,J. A., and HOFBAUER, A. (1977). SPREIJ, T. E. (1971). Cell death during the development of the imaginal disks of Calliphora erythroCell clones and pattern formation: on the lineage of cephala. Neth. J. Zool. 21, 221-264. photoreceptor cells in the compound eye of DroSTERN, C. (1969). Somatic recombination within the sophila. Wilhelm Roux Arch. 181,227-245. white locus of Drosophila melanoguster. Genetics CAMPOS-ORTEGA, J. A., JURGENS,G., and HOFBAUER, 62, 573-581. A. (1978). Clonal segregation and positional inforSULSTON,J. E., and HORVITZ, H. R. (1977). Postemmation in late ommatidial development in Drosophbryonic cell lineages of the nematode Caenorhubila. Nature 274, 584-586. ditis elegans. Develop. Biol. 66, 110-156. CARLSON, J. G. (1958). Microdissection studies of the dividing neuroblasts of the grasshopper Chorto- WADDINGTON, C. H., and PERRY, M. (1960). The ultrastructure of the developing eye of Drosophila. phugu uridifusciatu (De Gees). Chromosoma 5, Proc. Roy. Sot. London B 153, 155-178. 199-220.
there is no fixed lineage and emphasises the regulative nature of pattern formation in the eye. It may be of general interest that the exquisite retinal lattice can be constructed without determinative divisions, demonstrating that spatial mechanisms alone can be very precise. Defined cell lineage is found in the embryonic and postembryonic stages of many invertebrates. Examples are the postembryonic growth of nematodes (Sulston and Horvitz, 1977) and the formation of insect bristles (Lawrence, 1966). Even in these cases the cell divisions may be determinative not in their own right but more because they place the daughter cells appropriately (Lees and Waddington, 1942; Carlson, 1958; Sulston and Horvitz, 1977; Kimble and Hirsh, 1979).
BIOLOGY
71,142-152 (19%)
Cell Lineage in the Developing
Retina of Drosophila
A. LAWRENCE AND SHEILA M. GREEN of Molecular Biology, Hills Road, Cambridge, CB2 2QH, England
PETER MRC Laboratory
Received December 15, 1978; accepted January 31, 1979 Analysis of the cell lineage of the Drosophila retina is reported. Mitotic recombination within the white locus results in the formation of small red spots in white eyes; these are found under the dissecting microscope. The spot frequency is low (never more than l/30 eyes) so that there can be no doubt that each spot is a single clone. Eyes bearing a clone are serially sectioned and all retinula and all pigment cells scored as white or white’. We describe the constitution of 101 clones and examine the disposition of the marked cells in the retinal lattice. The clones are apparently random combinations of the marked cell types-for example, two-celled clones containing one pigment and one retinula cell are frequently found. Our results appear to rule out fixed cell lineage as a determinative mechanism in ommatidial development. INTRODUCTION
The intricate array of cells in the insect retina is so precise that it has been compared to a crystal (Benzer, 1973). In Drosophila the unit cell of this “crystal” consists of eight photoreceptive retinula cells, four cone cells, a four-celled bristle (Waddington and Perry, 1960), two primary pigment cells, and about four other pigment cells (Ready et al., 1976). The precision of this lattice led to the idea that it would develop by a precise cell lineage (Bernard, 1937; Kuhn, 1965). From studies with mosaic eyes, we now know that in Drosophila (Hanson et al., 1972), Oncopeltus (Shelton and Lawrence, 1974), and Periplaneta (Shelton et al., 1977) this mechanism does not operate: The cell lineages of ommatidia are indeterminate, each ommatidium descending from several precursor cells. These studies led to the conclusion that “cell determination within ommatidia is not connected with cell lineage but is dependent on cell position within the developing ommatidium” (Shelton and Lawrence, 1974). In Drosophila and the locust (Anderson, 1978) the formation of ommatidia is a gradual process, with certain cell types being generated before others-for example, in Drosophila, retinula cells 1, 6, and 7 142 0012-1606/79/070142-11$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
are added to preexisting clusters of cells at the end of eye development (Ready, 1973; Ready et al., 1976; Campos-Ortega and Gateff, 1976) so that in the development of the array “the final cell type may be determined according to the lattice position into which the cell is recruited” (Ready et a& 1976). It has been pointed out by Campos-Ortega that these studies do not exclude some lineage rules, which might operate in the last few cell divisions. For example, the developing retina could be subdivided into two sets of cells, one generating only pigment and one generating only retinula elements-the progeny of these cells would then assort themselves appropriately as the ommatidial clusters form (Campos-Ortega and Hofbauer, 1977). Lineage rules of this kind should be detectable in clones made during the last few cell divisions; one such approach has been employed (Campos-Ortega and Hofbauer, 1977; Campos-Ortega et al., 1978) but is subject to technical difficulties. Nevertheless, the authors concluded that their results “strongly suggest that fixed cell lineages are involved in the development of ommatidia.” Here we describe a new approach which obviates most of the technical problems.
LAWRENCE AND GREEN
Development of Ommatidia
143
optics. phase-contrast and bright-field Some clones were also examined in the electron microscope. Not all clones were successfully sectioned, the most frequent MATERIALS AND METHODS cause of failure being poor fixation-some We have used the method of Stern (1969) clones were lost because of this, especially of producing white+ clones in white eyes, during the early phase of our work. Screenby generating mitotic recombination within ing was done independently by the two the white locus. Two different white alleles authors-there was almost complete agreeare arranged in trans, such that mitotic ment (>90% of clones) when notes were recombination between them can form a compared. The few cases of disagreement wild-type chromosome (Fig. 1). w’ 0 were were resolved or, in two cases, discarded crossed to w65a256 and eggs collected and because we felt the preservation was inadirradiations of 1000 R performed in the equate. In many cases we were unable to usual way (Lawrence and Morata, 1977). distinguish between secondary and tertiary The time of irradiation was fixed in number pigment cells (Ready et al., 1976); in the of hours before puparium formation (hr tables we treat them as one cell type. BPF) (Garcia-Bellido and Merriam, 1971). Fl 0 were screened under the dissecting Assessment of Screening Efficiency microscope and clones containing one or The sections were examined for marked more pigment cells could be seen (but not pigment or retinula cells; all eight retinula those containing only retinula cells). The Rl-R8 (Dietrich, 1909) and all pigment first observer always missed some clones cells could be scored (Fig. 3 shows arrangeand therefore all flies were screened at least ment of the cells). When there were blocks twice and usually three times. The third of marked secondary and tertiary pigment observer still found a few clones so some cells, there was occasionally a problem in clones must have been missed. In one ex- determining the exact number markedperimental series 500 unirradiated controls deeper sections were helpful here, as the were included without the observers know- cells could be individually scored as discrete ing which flies had been irradiated-3 clusters of granules. The best region for clones were found in 1000 control eyes.’ scoring retinula cells R8 was at their most Eyes containing clones were fixed in half- distal extent and it was also helpful that strength Karnovsky (1965) fixative (see these cells had larger pigment granules than Ready et al., 1976) and embedded in aral- the other retinula cells (Rl-R7). After dite. The blocks were oriented so that the study of the selected clone, the remainder clone would be cut at right angles to the of the eye was carefully screened for other long axes of the ommatidia and serially clones. The fraction of each eye screened sectioned at 2 pm for observation using (usually 2/) was recorded, so that the sum ’ Stern’s purpose was to estimate the spontaneous total effectively searched for additional frequency of mitotic recombination. He found only 4 clones could be used below. In one series clones in 6137 flies, whereas our yield (3/500) is some (irradiation at 28 f 4 hr BPF) where the 10 times higher-the cause of the difference probably clone frequency detected under the dissectbeing the diffkulty of detecting very small clones. ing microscope was 1 clone per 30 eyes, 53 Counterbalancing this was the assumption (made by Stern in his calculations) that ommatidia had to be eyes were successfully sectioned and a entirely pigmented to be storable. There are six pigsummed area equivalent to 34 eyes was ment cells per ommatidium which if singly labelled screened. One eye had a second clone which can be detected, so that his calculated frequency of included a marked pigment cell, and two mitotic recombination (0.5-l/10”) was perhaps only a eyes contained additional clones of retinula slight underestimate.
Our results appear to rule out rigid cell lineages as a determinative mechanism in the development of ommatidial cell types.
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cells only. Of the other 119 eyes that were sectioned (irradiated at diverse ages >56 hr BPF), no clones that had been overlooked down the dissecting microscope were found, with the sole exception of one clone containing three retinula cells (irradiation at 39 + 1 hr BPF). We can conclude that (i) the screening for clones is efficient when they contain pigment cells and (ii) clones that were exclusively retinula cells were not detected down the dissecting microscope. RESULTS
VOLUME 71,1979
frequency and size should be reciprocally related, so that one would have expected the frequency at 28 + 4 hr BPF to be more than twice that at 52 + 4 hr BPF. In fact, it is only some 50% higher-probably because a much larger fraction of the clones produced at the later time consists of retinula cells only, and is therefore missed in the screening. The lower frequency and small size of clones found after irradiation at 4 f 4 hr BPF suggest that cell division in the eye has nearly ceased by that time. Position of Clones
Frequency and Size of Clones After pilot studies, certain ages of irradiation were chosen for detailed analysis, and the mean frequency and size of clones produced at these times are shown in Table 1. The log size of clones decreases as pupation approaches, until at 4 f 4 hr BPF most of the clones found are of one cell only. Clone
Direct observation (Becker, 1957; Ready et al., 1976) has shown that the eye develops in an orderly manner from posterior to anterior. By the middle of the third instar, ommatidial clusters can be detected at the posterior margin of the eye, and progressively, clusters appear more anteriorly. The process is completed shortly after pupation. w65a25 white
,65a25
we
red
FIG. 1. Mitotic
recombination
within
the white locus can generate a white’ TABLE
chromosome.
1
VARIATION OF CLONE FREQUENCY AND SIZE WITH AGE AT IRRADIATION No. of Age (hr BPF) No. of eyes” Frequency* Log size f SE clones” Unirradiated controls 3 loo0 0.003 4*4 19 1406 0.013 0.06 + 0.03 28 f4 89 2614 0.034 0.32 + 0.04 2736 0.022 0.73 + 0.07 59 52 + 4
Mean sizeb 1.2' 2.1 5.3
’ These numbers are based on clones found under the dissecting microscope, not all of which were successfully sectioned. b Size estimated from eyes successfully sectioned (Tables 2 and 3). 'N=14.
Development
LAWRENCE AND GREEN
ula cell precursors (Campos-Ortega and Hofbauer, 1977), small clones should contain either retinula cells or pigment cells, but not both. Table 2 gives the constitution of the clones found at this time, and it would appear that clones can be of many combinations, although because of the screening method, all contained at least one pigment cell. For example, clones 113, 123, 204,205, and 210 are of only two cells each, one pigment cell and one retinula cell. One might expect segregation between the polar retinula cells (R2-5) and the equatorial ones (Rl, 6, and 7), but this is not supported by the data (e.g., clones 120 and 230). Most small clones contained only one example of each retinula cell type, but two small clones showed that this is not a rule. One (made at 33 + 8 hr BPF) was of four cells, one primary and one secondary pigment cell and two retinula cells Rl, while another (195, made at 52 + 4 hr BPF) was of only four cells yet had two retinula cells R2 (Fig. 4e). Those clones produced at 52 f 4 hr BPF are on average larger (Table 3) and therefore not so ideal for revealing late lineage restrictions, but they are consistent with the earlier clones. They show no pattern and sometimes include several examples of the same cell type, even when other cell types are not represented (e.g., 196).
In all aspects the posterior part of the eye develops in advance of the more anterior parts, with waves of mitoses moving across the eye from posterior to anterior (Ready et al., 1976). Accordingly, one would expect clones induced at any one time during this process to vary in size depending on their position within the eye, anterior clones being larger; the positions and sizes of the clones were therefore plotted as accurately as possible on standard eye diagrams (Fig. 2). At 52 & 4 and 28 f 4 hr BPF the clones are distributed over most of the eye, and there is an approximate relationship between clone size and position. At 28 f 4 hr BPF the four largest clones (9, 10, 11, and 13) are found near the anterior margin, with the smaller clones being more common posteriorly. The most posterior part of the eye is free of clones. At 4 +- 4 hr BPF the few clones are mostly confined to the anterior part of the eye. Constitution of Clones Examples of the preparations are shown in Figs. 4 and 5. Many of the clones produced at 28 + 4 hr BPF and at later times were small, the largest being 13 cells. It is here that one might expect lineage restrictions to show; for example, if there were a segregation between pigment cell and retin424hBF’F
D
v
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of Ommatidia
28 t4hPPF
52+4hBF’F
D
D
v
v
FIG. 2. Distribution on eye of clones produced at three ages. The clones were originally plotted, by deadreckoning on a basis of ommatidial row coordinates, on a standard eye diagram. Each number represents the position of one clone containing that number of cells. A, anterior; P, posterior; D, dorsal; V, ventral.
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TABLE 2 CLONES PRODUCED BY IRRADIATION AT 28 f 4 HR BPF”
Clone No. 376 4ob
109 113 122 123 124 173 178 200 204 205 207 208 210 237 238 251 43b 107 114 175 231 65 106 203 230 73 176 201 71 172 206 120 177 74 234 250
Number cells 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 4 4 4 4 5 5 5 6 6 6 7
9 10 11 13 150
I
II + III
Rl
R6
R7
R2
R3
R4
R5
R.8
2 1 1 1
1 1 1 1 1
1
1 1 1 1
1 2 1 1 1 2 1 2 1 2 27
I 1 1 2 1 1 1 2 1 1
1
1 1
1 2 1 1 1 1 3 3 1 1 1 2 3 1 5 2 2 3 4 3 2 3 2 5 5 70
1 1 1
1 1
1
1 1
NS’
1 1 1 1 1
1 1 1
1
1
1
1 1 1 1
I
1 1 1 1 9
1 8
1 1
4
1 1 2 6
1 1 3
1 1 1 1 1 11
1 1
NS
1 8
1 4
DIn the same series 23 single-cell clones were found; of these, 8 were primary pigment cells (I) and 14 were secondary (II) or tertiary (III). There was one clone of 1 and one clone of 2 retinula cells in these eyes. ’ Irradiated 20 f 4 hr BPF. ’ Not storable.
It is known (Ready et al., 1976; CamposOrtega and Hofbauer, 1977) that only the pigment cells and retinula cells Rl, R6, and R7 are formed by the final mitotic front to sweep across the eye from posterior to anterior. The other retinula cells are formed in previous mitoses. One would therefore expect that clones would include labelled
Rl, R6, and R7 cells more often (CamposOrtega and Hofbauer, 1977). However, this is only true if a sufficient number of the clones is produced in cells in a stage after the formation of the R2-R5 and R8 cells but before or during the generation of the Rl, R6, and R7 cells. As only a small proportion of each irradiated eye is actually in
LAWRENCE AND GBEEN
147
Development of Ommatidia
TABLE
3
CLONES PRODUCED BY IRRADIATION AT 52 f 4 HR BPF Clone No. 131 220B 243 247 69 216 222 223 242 244 130B 184 185 188 195 212 224 198’ 246 197 215 183 171 217 130A 182 245 241 189 214 196 220A 186 190
-
Number cells 2 2 2 2 3 3 3 3 3 3 4 4 4 4 4 4 4 6 6 8 8 9 10 10 11 11 14 16 19 19 23 29 32 55 340
II + III
I
Rl
R7
R6
R2
R3
___~ R4
R5
R8
2 1
1
1 1 1 1 1 2 2 2
1 1
1 2 1 1 1 2 1 3 1 2 2 2 3 1 1 2 3 2 3 5 4 9 61
1 2 1 1 5 2 1 2 2 2 5 3 7 8 5 9 7 12 7 10 15 122
1
1 1 1
1 1
1 1 1 2
1
20
-
1
1
1
2
1 1 1 2 1 2 2 4 18
1 1
1 2 1 1 1 3 2 16
1
1
1 1
1
1 1 1 2 2 1 1 2 2 3 5
1 1 2
2 1 3 1 5 17
a In the same series four single-cell clones of primary (I), secondary (II), or tertiary (III) pigment cells were found; there were no other clones. Two pairs of clones (130A, 130B; 220A, 220B) were each found in one eye. * On extreme edge of eye.
this stage, one might not expect the preferential labelling of Rl, R6, and R7 to be detectable (Tables 2 and 3). The border of large clones, or patches in gynandromorphs (Benzer, 1973; Ready et al., 1976), is very uneven, showing mosaicism spread over about two ommatidial rows. It is therefore no surprise that the small clones are made up of somewhat scattered cells. Usually the clones are confined to a patch consisting of a central plus the six adjacent ommatidia, but within this area they can be well separated. Some examples
are shown in Fig. 3, while a series of sections of one clone (183) is illustrated in Figs. 4ad. We have found five clones at the extreme edge of the eye. These were not included in the tables, as they, with one exception (Fig. 4f), contained only peripheral pigment cells. They confirm (Ready et al., 1976) that the peripheral ommatidia are surrounded by a continuous sheath of pigment cells. Roughening X irradiation
produces a band of rough-
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DEVELOPMENTAL BIOLOGY
LEFT EYE DORSAL
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RIGHT EYE DORSAL
FIG. 3. Examples of 10 clones, to show the distribution of marked cells (shown in black) in the lattice. The position of the clone in the eye (right R or left L, dorsal D or ventral V) is given as well as the clone number (see Tables 2 and 3). Orientation sketches to show numbering and nomenclature (from Ready et al., 1976) are given at the top. 1-8, Retinula cells; HNG, hair nerve group; I, II, and III, primary, secondary, and tertiary pigment cells; A, anterior; P, posterior. Clones 186 and 123 are at the edge of the eye.
FIG. 4. (a-d) Sections of clone 183 (compare Fig. 31 at four different levels. (a) Level of the primary pigme bnt cells; the two marked primaries (I), a secondary (II), and a tertiary (III) are seen. (b) At the most distal part Of the retinula cells, one marked retinula cell R4 is arrowed. (c) Lower in the retinula cells; solid arrows point to marked retinula cells, and open arrows to unmarked ones. In white’ retinula cells the pigment granules Ewe found clustered at the base of the rhabdomere and scattered elsewhere in the cell. (d) At the level of the 1K8 cells, one is labelled. The marked secondary and tertiary pigment cells can be traced through all four sectioi 1s. (e) Clone 195 (Table 3) showing two labelled retinula cells R2 and one H3. (0 Section through clone 198 (Tat de 3) at extreme edge of eye; this clone marks five secondary pigment cells and one primary pigment cell (not in plane of section). Sections ca. 2 pm; phase contrast. x 1200. 149
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DEVELOPMENTAL BIOLOGY
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FIG. 5. Electron micrograph of section through clone 65 (Table 2). Large pigment granules are present in the marked secondary pigment cell (II), and smaller ones (p) in the marked retinula cells R4 and R5. Large numbers indicate the rhabdomeres, and smaller ones the cytoplasm of the retinula cells RI-R7. Arrows indicate attachment desmosomes between neighbouring retinula cells.
LAWRENCE AND GREEN
ened ommatidia parallel to the dorsoventral axis, its position moving anteriorly with later irradiations (Becker, 1957). This roughening is associated with defective ommatidia which lack one or more cells (Campos-Ortega and Hofbauer, 1977). In our hands, the roughening was variable. In one case only, marked cells were found in a defective ommatidium, but in other cases roughened ommatidia were found near to the clone (one is shown in Fig. 3, 205). If rough ommatidia were plotted in relation to the clone and different eyes compared, it was found that there was no simple spatial relationship. For example, the band of roughening, which could affect as many as 20 ommatidia in some eyes, was usually posterior to the clone, but rough ommatidia were also found near the clone and anterior to it. DISCUSSION
Small red spots are generated in the developing white retina of Drosophila, and their size and constitution recorded. Because the spots are rare (never more than 1 spot/30 eyes), each spot must be an individual clone. Even when produced at the very end of retinal development, the clones show no evidence for rigid cell lineages, and label the pigment and retinula cells in diverse combinations. These results support earlier views of ommatidia development (Ready et al., 1976; Shelton and Lawrence, 1974) which envisage local cell migration and an orderly assembly of uncommitted cells into the lattice. Rigid cell lineages in the development of ommatidia have been proposed by ComposOrtega and colleagues after an extensive series of experiments (Hofbauer and Campos-Ortega, 1976; Campos-Ortega and Hofbauer, 1977; Campos-Ortega et al., 1978). These authors made clones of recessive markers such as white and rdgB in heterozygous eyes of wild-type phenotype. One problem was that with white clones there was sometimes difficulty in scoring the lack of pigment in the retinula cells (CamposOrtega and Hofbauer, 1977). Also, because
Development
of
Ommatidia
151
of the large number of spots found per eye, an operational criterion had to be devised to define when a spot should be treated as a clone; this was done when marked cells were confined to a group of seven ommatidia (a central one and its six immediate neighbours). This operational criterion is suspect: For example, Fig. 3 shows one instance (197) which would have been classified as two clones (Campos-Ortega and Hofbauer did not score primary pigment cells). Further, evidence that some of the spots which do meet the criterion are nevertheless two clones is presented by CamposOrtega et al. (1978). These authors conclude that double hits could explain all cases of “clones” containing both retinula and pigment cells. However, we have several cases where small clones do contain both types of cell (Tables 2 and 3) and no suggestion that there is any segregation. The clearest examples are those clones of only two cells, one retinula and one pigment cell (40,113,123,204,205,210,237, and 238, Table 2; 220B, 243, and 247, Table 3). Their operational criterion may also have led these authors to occasionally classify two clones as one, and these problems may well be the root cause of the many discrepancies between their results and ours. The assessment of cell lineage by clonal analysis requires certain discrimination between single clones and double events; without this discrimination, conclusions can be insecure. We may not have enough clones to test all possible lineage rules, but Tables 2 and 3 list sufficient to exclude all simple ones. Our results would appear to rule out cell lineage as a determinative mechanism in the development of ommatidia. This conclusion refers only to those cell types we were able to score (the hair nerve groups and cone cells were not scored). Ommatidial assembly is probably normally associated with some cell death (Spreij, 1971), and no doubt the X irradiation kills many additional cells. Nevertheless, apart from a few roughened ommatidia, the pattern is perfect-an observation which supports our view that
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DIETRICH, W. (1909). Die Facettenaugen der Dipteran. Z. Wiss. Zool. 92,465-539. GARCIA-BELLIDO,A., and MERRIAM, J. (1971). Parameters of the wing imaginal disc development of Drosophila melanogaster. Develop. Biol. 24, 61-87. HANSON,T. E., READY, D. F., and BENZER, S. (1972). Use of mosaics in the analysis of pattern formation in the retina of Drosophila. Caltech Biol. Annu. Rep. 40. HOFBAUER, A., and CAMPOS-ORTEGA,J. A. (1976). Cell clones and pattern formation: genetic eye mosaics in Drosophila melanogaster. Wilhelm Roux Arch. 179, 275-289. KARNOVSKY, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27, 137A-138A. KIMBLE, J. E., and HIRSH, D. (1979). The postembryonic cell lineages of the hermaphrodite and male gonads in C. eleguns. Develop. Biol. (in press). KUHN, A. (1965). “Vorlesungen iiber Entwicklungsphysiologie.” Springer-Verlag, Berlin. LAWRENCE,P. A. (1966). Development and determination of hairs and bristles in the milkweed bug Oncopeltus fasciutus, Dall. J. Exp. Biol. 44, 507We thank Jose Campos-Ortega for advice on tech522. niques and discussion; Jonathon Hodgkin, Judith Kimble, and John Sulston for help with the manu- LAWRENCE, P. A., and MORATA, G. (1977). The early development of mesothoracic compartments in Droscript; Gary Struhl and John White for encouragesophila. An analysis of cell lineage and fate mapping ment; and Paul Johnston for assistance with the and an assessment of methods. Develop. Biol. 56, screening. 40-51. REFERENCES LEES, A. D., and WADDINGTON, C. H. (1942). The ANDERSON,H. (1978). Postembryonic development of development of bristles in normal and some mutant the visual system of the locust, Schistocerca gretypes of Drosophila melanoguster. Proc. Roy. Sot. garia. I. Patterns of growth and developmental London B 131,87-110. interactions in the retina and optic lobe. J. Embtyol. READY, D. F. (1973). Pattern formation of the retina Exp. Morphol. 45, 55-83. in Drosophila. Caltech Biol. Annu. Rep. 203. BECKER, H. J. (1957). Uber Rontgenmosaikflecken READY, D. F., HANSON, T. E., and BENZER, S. (1976). und Defektmutationen am Auge von Drosophila Development of the Drosophila retina, a neurocrysund die Entwicklungsphysiologie des Auges. Z. Intalline lattice. Develop. Biol. 53, 217-240. dukt Abst. Vererb. 88, 333-373. SHELTON, P. M. J., ANDERSON, H. J., and ELEY, S. BENZER, S. (1973). Genetic dissection of behavior. Sci. (1977). Cell lineage and cell determination in the Amer. 229,24-37. developing compound eye of the cockroach, PeriBERNARD, F. (1937). Recherches sur la mophogenie plunetu americana. J. Embryo1 Exp. Morphol. 39, des yeux composes d’arthropodes. Bull. Biol. Fr. 235-252. Belg. Suppl. 23. SHELTON, P. M. J., and LAWRENCE, P. A. (1974). CAMPOS-ORTEGA,J. A., and GATEFF, E. (1976). The Structure and development of ommatidia in Oncodevelopment of ommatidial patterning in metamorpeltus fasciutus. J. Embryol. Exp. Morphol. 32, phosed eye imaginal disc implants of Drosophila 337-353. melanogaster. Wilhelm Roux Arch. 179, 373-392. CAMPOS-ORTEGA,J. A., and HOFBAUER, A. (1977). SPREIJ, T. E. (1971). Cell death during the development of the imaginal disks of Calliphora erythroCell clones and pattern formation: on the lineage of cephala. Neth. J. Zool. 21, 221-264. photoreceptor cells in the compound eye of DroSTERN, C. (1969). Somatic recombination within the sophila. Wilhelm Roux Arch. 181,227-245. white locus of Drosophila melanoguster. Genetics CAMPOS-ORTEGA, J. A., JURGENS,G., and HOFBAUER, 62, 573-581. A. (1978). Clonal segregation and positional inforSULSTON,J. E., and HORVITZ, H. R. (1977). Postemmation in late ommatidial development in Drosophbryonic cell lineages of the nematode Caenorhubila. Nature 274, 584-586. ditis elegans. Develop. Biol. 66, 110-156. CARLSON, J. G. (1958). Microdissection studies of the dividing neuroblasts of the grasshopper Chorto- WADDINGTON, C. H., and PERRY, M. (1960). The ultrastructure of the developing eye of Drosophila. phugu uridifusciatu (De Gees). Chromosoma 5, Proc. Roy. Sot. London B 153, 155-178. 199-220.
there is no fixed lineage and emphasises the regulative nature of pattern formation in the eye. It may be of general interest that the exquisite retinal lattice can be constructed without determinative divisions, demonstrating that spatial mechanisms alone can be very precise. Defined cell lineage is found in the embryonic and postembryonic stages of many invertebrates. Examples are the postembryonic growth of nematodes (Sulston and Horvitz, 1977) and the formation of insect bristles (Lawrence, 1966). Even in these cases the cell divisions may be determinative not in their own right but more because they place the daughter cells appropriately (Lees and Waddington, 1942; Carlson, 1958; Sulston and Horvitz, 1977; Kimble and Hirsh, 1979).
