Cell lineage and development in the larval epidermis ofDrosophila melanogaster

DEVELOPMENTAL

BIOLOGY

Cell Lineage

73, 256-271 (1979)

and Development in the Larval Epidermis melanogaster

JANOS SZABAD,* Zoologisches

TRUDI

Znstitut der Universittit *Biological Research Received

February

of Drosophila

SCH~~PBACH, AND ERIC WIESCHAUS'

Ziirich, Center,

Kiinstlergasse IS, CH-8006 P.O.B. 521, H-6701 Szeged,

12, 1979; accepted

in revised

form

May

Ziirich, Switzerland, Hungary

and

14, 1979

Larval and adult gynandromorphs were produced to study the origin and development of the larval epidermis in Drosophila. In mosaic larvae the genetic marker mal was used to score the genotype of all the cells in the epidermis and thus to follow the gynandromorphic border through this large continuous sheet of cells. This mosaic border proved to be rather convoluted, such that even small areas of 46 cells were mosaic in 23% of the cases. From the similarity of this value to the frequency of mosaicism in the adult legs, it was concluded that the epidermis of the larval segments Tl-A7 (thoracic-abdominal) arises from about 2000 blastoderm cells, thus requiring two rounds of cell division to achieve the 7500 cells present at final differentiation of the larval epidermis. An increased frequency of mosaic borders was observed at the ventral midline of the larvae, indicating the “loss” of 16% of the cells in the circumference of the blastoderm which invaginate ventrally during gastrulation. A smaller increase in frequency of borders (4.8%) was found on the dorsal midline, reflecting the formation of the extraembryonic membranes. Finally, an increase in frequency of mosaic borders was observed on all segment boundaries, which can be interpreted as the result of cell lineage restrictions between the larval epidermal cells of adjacent segments arising at the blastoderm stage. In all mosaic larvae, the genotype of the six imaginal histoblast clusters per segment closely corresponded to the genotype of the surrounding larval cells. The imaginal histoblasts thus originate as clusters of cells within the large continuous sheet of cells giving rise to the larval epidermis.

are well suited for developmental studies. Each possesses a well-defined Gattern of During its lifetime, Drosophila builds cuticular structures which allows identifytwo different integuments. The first of ing individual subregions within the intethese, the larval epidermis, is completed gument. Mutations are known which alter during embryonic development. Although both the larval and the adult patterns either the animal makes a new cuticle with each (bithorax, Polycomb; Lewis, moult, the underlying larval epidermal cells zygotically 1978) or maternally (bicaudut; NiiBleinremain the same throughout larval develVolhard and Gehring, personal communiopment and the pattern of the cuticle uncation), indicating that both types of epidergoes only slight modifications with each dermal cells share certain developmental new larval instar. At metamorphosis the mechanisms in common. The relationship larval cells are histolysed and the adult between the adult and larval primordia, epidermis is formed by the fusion of the however, remains poorly understood. We imaginal discs and histoblasts specific for do not know, for example, whether the cells different regions of the adult. which give rise to corresponding regions of Both the larval and the adult epidermises the larval and adult epidermis are derived from the same regions of the embryo, ’ Present address: European Molecular Biology whether these cells undergo the same exLaboratory Postfach 10.2209, D-69 Heidelberg, Germany. tent of proliferation during embryonic de256 INTRODUCTION

0012-1606/79/120256-16$02.00/O Copyright All rights

0 1979 by Academic Press, Inc. of reproduction in any form reserved.

SZABAD,

SCH~~PBACH,

AND

WIESCHAUS

velopment, and whether they undergo comparable developmental restrictions. Most of what is known about the development of the larval epidermis derives from 1937, morphological studies (Poulson, 1950). The embryonic development of the adult epidermis, on the other hand, has been extensively st,udied using genetic mosaics produced by the loss of an X chromosome and by X ray-induced mitotic recombination. In the experiments described below, we have produced mosaics for the larval epidermis using the loss of an unstable ring X chromosome during early cleavage. Since the genotype of the histoblast cells which give rise to the adult abdomen could be identified in the same preparations, it was possible to study not only the development of the larval epidermis but also its relation to the precursors for the adult epidermis. MATERIALS

AND

METHODS

Gynandromorphs were produced by crossing females homozygous for the genetic markers y u f mat to males carrying the unstable ring X chromosome In (1) w“’ and a marked Y chromosome (Y-y’). (For a description of all mutations, see Lindsley and Grell (1968); for In (1) w”‘, see also Hinton (1955); Hall et al. (1976).) The gynandromorphs thus consisted in part of female (XX) cells which were phenotypically wild type and in part of maIe (X0) cells marked withy f mal. Since ma1 hemizygous cells lack aldehyde oxidase activity, the genotpye of the internal cells can be identified after staining the tissue histochemitally for aldehyde oxidase activity (Janning, 1972, 1976; Kuhn and Cunningham, 1978; for further details of the procedure, see below). This histochemical staining results in a dark blue coloration of mal+ cells, whereas mal cells remain unstained. The progeny of the gynandromorph producing cross were raised at room temperature on standard Drosophila medium. Third-instar larvae were collected as they

Larval

Epidermal

Mosaics

257

left the food and fixed and stained as described below. Part of the progeny from the same parents was also allowed to develop to the adult stage. These adult flies were carefully inspected under a dissecting microscope and all apparent gynandromorphs were prepared as described by Szabad (1978) for analysis under a compound microscope. The histochemical staining of the larval epidermis proceeded by washing the thirdinstar larvae with 70% ethanol and distilled water, etherizing the well-dried larvae, and injecting them with 1% ice-cold glutaraldehyde in Ringers’ until they assumed a fully streched “blown-up” appearance. After a fixation period of 0.5 hr at O”C, the posterior, or in some cases the anterior, end of the larvae was cut open in cold Ringer’s solution (buffered at pH 7) and the whole larvae were turned inside out via this anterior or posterior opening. Most of the internal larval organs were removed. The larval epidermis with the directly underlying musculature was then stained for aldehyde oxidase activity (Janning, 1976). By this technique the genotype of the cells in the larval epidermis became apparent, including both larval cells and the cells within the clusters of imaginal histoblasts. The larval oenocytes which sit as clusters of about six cells on the lateral midline in each abdominal segment could also be scored for the presence or absence of aldehyde-oxidase activity. The larval musculature did not show any specific aldehyde oxidase activity in maZ+ larvae, although the fibres often picked up a light blue coloring due to their proximity to the maZ+ epidermis. The nonspecificity of this staining is indicated by the fact that it was never observed in muscles of wild-type larvae which were removed from the larval hypoderm after fixation and stained separately for aldehyde oxidase activity. The stained larval epidermal preparations were examined under a dissecting microscope, and each epidermis classified as

258

DEVELOPMENTAL

BIOLOGY

VOLUME

73,197s

mosaic was cut longitudinally, opened out, (4) Following standard gynandromorph and mounted in water-soluble Faure’s procedures (Hotta and Benzer, 1973), sturt mounting solution for inspection under a distances were calculated between different compound microscope. The mosaic pattern regions of the larval epidermis, as well as of each larval epidermis was drawn indebetween specific landmarks of the larval pendently by at least two of the authors and adult epidermis. onto a standard grid whereby the segment Sturt distances between the histoblast boundaries, the ventral setae, and in particnests, as well as the distances between speular the pattern and attachment sites of the cific adult landmarks (i.e., individual terlarval musculature served as reference gites and sternites), were not calculated points (Fig. lb). In some preparations the from the drawings, but instead by restoring muscle attachment sites or the regions unthe preparations. derlying the ventral musculature were RESULTS poorly stained, although judging from the General characterization of the larval surrounding cells, they were most probably gynandromorphs. Gynandromorphs were mal+. In all questionable cases the mosaic produced by mating y v f maZ?O to In (1) border was drawn with minimal convoluwvcs$. A total of 2231 larvae was stained tions (as a straight line). The drawings were used in a number of further analyses: for aldehyde oxidase activity, and when (1) The relative size of the female and their epidermis was examined, 152 mosaics male patches was measured using standard (6.8%) were detected (Table 1). This is planimetric techniques. smaller than the yield of adult cuticular (2) The frequencies with which the mogynandromorphs from the same cross saic border line cut through different re- (9.1%). This difference is probably due to gions of the epidermis were determined by the fact that the larval preparations could dividing circumferential lines (the segment only be scored in the thorax and first through seventh abdominal segments (Alboundaries) and longitudinal lines (dorsal A7), whereas among the adults, gynandroand ventral midline and the two lateral midlines) into subsections (Fig. 1). The av- morphs could be detected even when they were mosaic only in the head or genitalia. erage width of each subsection was two larval epidermal cell diameters. In this way, Among the adults, 0.8 and 0.5% were gyneach segment boundary was divided into 24 andromorphs of this type. With the undesubsections and each longitudinal line into tected head and terminal gynandromorphs, 5 subsections per segment. Each individual we estimate the total gynandromorph popsubsection was checked in the drawings of ulation among the larvae to be 8.1%. In all mosaic preparations and the number of comparison with an adult population, the gynandromorphs in which the mosaic borlarval values, which are based only on the der cut through this subsection was re- thorax and abdomen, should be multiplied by 6.8/8.1 (= 0.84). This correction factor corded. (3) The frequency with which areas of makes very little difference for most of the considerations in this paper and all data different size on the larval epidermis were mosaic was measured by placing circles of will initially be given in terms of the movarying diameters on the drawings and saics actually detected (see, however, p. counting the number of cases in which the 270). mosaic border cut into these circles. The Microphotographs of three of the larval circles were never placed on the ventral or epidermal gynandromorphs are shown in dorsal midline, and segment boundaries Fig. 2. Of the 152 gynandromorphs, 12 were were also avoided, unless the size of the damaged too severely during the histological procedure to allow detailed analysis. circle made this impossible.

-

t . _

- :!;

. : -.

.

FIG. 1. Semi-schematic illustration of the larval epidermis and the grids used in drawing the mosaic patterns. Figure la gives the general morphology in the midabdominal (A3-A5) region of the larva. The upper segment shows the pattern of the muscles as viewed from the inside. In the second segment, the positions of the ventral histoblasts (v), anterior dorsal histoblasts (ad), posterior dorsal histoblasts (pd), and oenocytes (oe) are given relative to certain muscles lying close to the epidermis. The third segment shows the pattern of setae and sensory elements in the larval cuticle. In the ventral setae (vs), the first and fourth rows point anteriorly (LohsSchardin et al., 1979) and mark the attachment sites for two of the three sets of muscles which bind near the ventral midline. In our drawings, the segment boundary was defined by the major line of muscle attachment sites and is shifted slightly posterior in the dorsal region of the animal. This means that in both the ventral and the dorsal cuticles, the anteriormost rows of setae would mark the posterior rim of the previous segment. Figure lb shows two segments of the grid onto which the mosaic patterns were drawn. The upper segment shows the major landmarks used for orientation of the drawing, the lower segment shows the subsections used to determine mosaic border frequencies in the anterior-posterior and circumferential directions and some of the differently sized areas used to determine the frequencies of mosaicism (see text). The areas depicted would represent approximately 5,26,46, and 62 larval epidermal cells, respectively. 259

260

DEVELOPMENTAL

BIOLOGY

The mosaic patterns of the remaining 140 were drawn onto blank forms. Figure 3 shows 48 of these drawings. Similar to the adult gynandromorphs previously described in the literature (Garcia-Bellido and Merriam, 1969; Hotta and Benzer, 1972), the mosaic patterns in the larval epidermis suggest that the chromosome loss occurs primarily in the first cleavage division. The male patch occupied, on an average, 45.7 + 23.8% of the larval epidermis. The variability with which the male and female genotypes were represented in different mosaics was, however, extreme; in 9 and 3% of the larval gynandromorphs, less than 10% or more than 90% of the total surface was male (Fig. 4). The most frequent mosaic pattern consisted of one male and one female patch. Cases with two male or two female patches were found with a frequency of 18%, and mosaics with more patches were rare (~1.4%). Each mosaic pattern in the 140 larval gynandromorphs was different from all others. The most striking difference from mosaic patterns observed in adult gynandromorphs is that the mosaic patterns in the larval epidermis are much more convoluted and the borders more jagged. Distribution of the mosaic border. In Drosophila embryos the initial cleavage plane is random (Parks, 1936) and therefore the probability of the mosaic border falling between adjacent blastoderm cells should be the same in all parts of the embryo (Hotta and Benzer, 1973; Schiipbach et al., 1978). Nonrandomness in the differentiated epidermis must be the product of later events in development, for example, the invaginations beginning at gastrulation. In order to detect deviations from random-

VOLUME

73, 1979 TABLE

1

NUMBER AND GENOTYPE OF LARVAE AND FLIES RECOVERED FROM THE CROSS y u f mal/y u f ma1 x zn(l)w”c/Y.y+” Genotype y v f mal/Y.y+ y v f mal/O y v f mal/Zn(l)w”C Gynandromorphs Total

Larvae I

1659 420 152 2231

Adults 5816 793h 1639 827 9075

n The classification was based solely on the genotype of the epidermis. In the larvae, the marker mal was used; in the adults, y and f. b This figure included 27 individuals which had y f cuticle but v+ eyes and evidently were internal “cryptic” mosaics with v+ tissue in the fat body or Malphgian tubules (Nissani, 1975).

ness, the circumference as well as the longitudinal axis of the larval epidermis were divided into equally sized subsections of two cell diameters in width and the frequency with which each subsection was cut by the mosaic border was determined. The circumference was divided into 24 subsections. Significant increases in border frequencies were found in the subsections at the ventral and dorsal midline (Fig. 5). 25.2% of all borders cutting through a given circumference fall into the three subsections at the ventral midline. By contrast, the fraction of the borders cutting three subsections in the lateral region of the epidermis was only 9.6% (3.2% per subsection) and thus the peak at the ventral midline represents a surplus of 15.6%. A considerably smaller peak (8.1%) is found lying directly over the dorsal midline. It represents slightly more than double the amount of borders expected from the average, such that the excess amounts to 4.9% of all borders. The tendency of the mosaic border to follow the ventral and dorsal midline is also

FIG. 2. Mosaic patterns in the epidermis of larval gynandromorphs. The mal’ regions (XX) are indicated by their dark staining for aldehyde oxidase activity, in contrast to the mal regions (X0), which remained colorless. The larva depicted in a has been opened up near its right side, the larva in b at its ventral midline, and the larva in c along its dorsal midline. The arrows indicate the position of the ventral midlines. Figure 2d is an enlargement of the ventral region of larva c (segments A4 and A5) where the mosaic border runs in a fairly straight line in the anterior-posterior direction, somewhat to the left of the midline as defined by the muscle attachment sites.

262

DEVELOPMENTAL

BIOLOGY

VOLUME

73,1979

FIG. 3. Distribution of XX and X0 cells in the epidermis of 48 larval gynandromorphs. The larvae have been drawn as though they had all been opened along their right sides. Although the mosaic border is often rather convoluted, most larvae possess one continuous male patch (uncolored) and one continuous female patch (darkly colored). Examples of larvae with more than one patch are the fourth and fifth larvae in the second and sixth rows.

apparent on examination of the patterns in individual mosaics depicted in Figs. 2 and 3.

Figure 6 plots the number of cases in which the XX/X0 border crossed subsections positioned along the anterior-poste-

SZABAD,

PfRCfNlAEf

SCH~~PBACH,

AND

Larval

WIESCHAUS

YAlflfSS

FIG. 4. The fraction of larval gynandromorphs which had male patches of different sizes. The size of the male patch was determined planimetrically in the drawings of 140 larval gynandromorphs and was expressed as the percentage of the total surface which was male.

rior axis of the larva. A series of small peaks was found directly overlying the segment borders. The XX/X0 borders fall 2.4 times as frequently into the subsections overlying the segment borders as into the two adjacent subsections to the side of each peak. Within the accuracy of our measurements, no other accumulation of borders could be discerned within segments T3-A7. When the regions on the ventral midline, on the dorsal midline, and overlying the segment boundaries are excluded from the analysis, the mosaic border cut equally frequently through subsections in the anterior-posterior and in the circumferential directions. Anterior-posterior subsections were cut with a probability of 4.3% (969 borders in 22,400 subsections). In the circumferential direction, they were cut with a frequency of 4.6% (1297 borders in 28,000 subsections).

Epidermal

263

Mosaics

possessesthree pairs of histoblast neststhe dorsal anterior, the dorsal posterior, and the ventral nests (Madhavan and Schneiderman, 1977; Lawrence et al., 1978). In our preparations, the numbers of histoblast cells in these nests are 11-13,6-g, and 12-16, respectively. Clonal analyses and cell counts indicate that histoblast cells are mitotically inactive during larval development (Garcia-Bellido and Merriam, 1971b; Guerra et al., 1973; Madhavan and Schneiderman, 1977). The number and arrangements of the histoblasts of third-ins&r larvae should therefore be representative of the situation at the end of embryogenesis. A very close cell lineage relationship between the histoblasts and the adjacent larval epidermis is indicated by the observation that the genotype of the histoblast nests always corresponds to the region of the larval epidermis in which they are situated. No case was found among the 5796 histoblast nests analysed where the histoblasts were of one genotype and the surrounding larval epidermis was of the other. In cases where the histoblast cluster was mosaic, the XX/X0 border ran continu-

I

YfNlnAl

DORSAL YlDLlNf

YIDlIWf

Relationship between the adult and larval abdomens. The integument of the adult abdomen consists of a dorsally situated tergite and a ventrally situated sternite. The precursors of these structures can be identified in the larva as dorsal and ventral nests of small histoblast cells imbedded among the polytenic epidermal cells of abdominal segments Al-A7. Each segment

1

larval

FIG.

5. Distribution

circumference

of mosaic

borders

circumference of 140 larval gynandromorphs.

along

the

The cir-

cumference was divided into 24 subsections (see Fig. 1) and the number of tunes each subsection was mosaic was determined. The figure represents the summed data for subsections on the anterior margins of the abdominal segments Al-A7.

DEVELOPMENTAL BIOLOGY

264

VOLUME 73,1979

7 13

52

1

89

j

133

132

40

n

THORAX FIG. 6. Distribution of mosaic borders along the anterior-posterior axis of 140 larval gynandromorphs. The 11 segments (Tl-A8) were divided into 55 subsections such that each segment boundary fell into the middle of one subsection (see Fig. 1). The number of times each subsection was mosaic was determined on the ventral and dorsal midline and the two lateral sidelines. The figure represents the summed data of the subsections on the four axes. The values in the anterior and posterior regions of the larva (stippled) are probably to some extent underestimates due to preparation damage.

ously between the histoblast and the adjacent larval epidermis cells (Fig. 7). Fate maps of the larval and adult abdomens were constructed from the data derived from larval and adult gynandromorphs (Fig. 8). Distances between adjacent tergites, between histoblast nests of adjacent segments, as well as between homologous points in adjacent larval segments were all about 10 sturts. Distances between tergites and sternites or dorsal and ventral histoblast nests within the same segment were larger (20 sturts), although still somewhat smaller than the distances between homologous structures on the right and left sides within the same segment (30 sturts). One discrepancy between the larval and adult data which we cannot explain was found in the frequency of mosaicism of adult tergites and dorsal histoblasts. In the adult, the frequency of mosaicism of hemitergites was uniform for segments A2-A6, with a mean of 15.4% (Table 2). The hemitergite of the first abdominal segment was more often mosaic (26.3%). In the larval

epidermis, in contrast, the frequency of mosaicism of the entire dorsal histoblast cluster (anterior and posterior nests considered as one cluster) amounted to only 8.8% and was the same in all segments (Table 2). It is possible that this lower frequency reflects a slight nonautonomy of our aldehyde oxidase staining, although the sharp border between staining and nonstaining cells in a mosaic histoblast would argue against this. Alternatively, the problem may relate to our uncertainty about the exact relationship between the larval precursors and the adult tergites. In addition to the dorsal and ventral histoblasts, a fraction of the adult abdomen near the tracheal opening is formed by the larval perispiracular cells (Robertson, 1936; Roseland and Schneidermann, 1979). Although these cells normally do not participate in the formation of the adjacent tergites (Lawrence et cd., 1978), it is possible that the higher frequency of tergite mosaicism we have detected in our adult gynandromorphs might represent occasional contributions of the perispiracular

SZABAD,

FIG. 7. Mosaic histoblast cluster histoblasts = pd.

SCHL~PBACH,

AND

WIESCHAUS

Epidermal

Mosaics

265

histoblast clusters in larval gynandromorphs. (a) Mosaic ventral histoblast cluster; (b) dorsal which is mosaic in the posterior nest. Anterior dorsal histoblasts = ad; posterior dorsal

cells to the ventral

margin of the tergite. of precursor cells. Cell counts in the larval epidermis showed that a larval segment, on average, contains some 650 larval cells. An additional 60 cells per segment were found in the imaginal histoblast nests of segments Al through A7. Thus the total number of cells making up the larval epidermis (11 segments) is about 7500. Since the ectodermal precursors divide between the blastoderm stage and final differentiation (Poulson, 1950; Madhavan and Schneiderman, 1977), the larval epidermis must arise from a smaller number of precursor cells. To estimate the number of precursor cells, the frequency of mosaicism of round-shaped areas on the larval epidermis was measured. These frequencies were then compared to those of imaginal structures where detailed information about the number of precursor cells is available from clonal analyses. Epidermal areas consisting of about 46 cells were mosaic with a frequency of 23.5%. In the population of gynandromorphs which had been allowed to develop to adults, individual legs were mosaic in 22-25s of the cases (Table 2). The similarity between this value and that found for an area of 46 larval cells indicates that the precursor cells for both types of structures are drawn from areas of equal

The

Larval

size at the blastoderm

stage.

number

DISCUSSION

The mosaic border in gynandromorphs at the blastoderm stage. The mosaic border which defines the male patch in gynandromorphs at the blastoderm stage will be more or less convoluted, depending on the extent to which the nuclei mix with each other during later cleavage divisions and during their migration into the egg cortex. Recent attempts to estimate the extent of convolution at the blastoderm stage have been based on the additiveness of sturt distances measured over long and short ranges (Janning, 1978) or on the comparison of relatively small sturt distances with the size of clones marking the progeny of individual cells at the blastoderm stage (Wieschaus, 1978). The former approach yielded values of 2.2 sturts per cell, the latter, 5.5. Other estimates based on the sturt distance across cell lineage restrictions thought to arise at the blastoderm stage yield values between 5 and 10 sturts (Lawrence and Morata, 1977; Wieschaus, 1978). These disparities lead to considerable differences in the interpretation of fate maps constructed from gynandromorph data. The discrepancy would be resolvable if the mosaic border could be followed at

266

DEVELOPMENTAL

4 ve.ntral

anterior

BIOLOGY

dorsal I

. if

32.4

’

I

21.5

k cl 1)

J

3$.2

FIG. 8. Summaries of the fate map data for the abdomen, derived from larval and adult gynandromorphs. In the larval map (a) sturt distances have been calculated between ventral histoblast clusters (V), dorsal anterior clusters (aD) and dorsal posterior clusters (pD). Sturt distances were also calculated between segment eights; these values are given in the margin of a. In the fate map of the adult abdomen (b), sturt distances were calculated between tergites and sternites. The data are derived from 140 larval and 631 adult gynandromorphs and represent the average values of all abdominal segments (Al-A7 for larval map, A2-A6 for adult map).

FREQUENCIES

VOLUME

73, 1979

the blastoderm stage itself. This is, unfortunately, not possible with the genetic markers and staining techniques used to distinguish male and female cells at later stages. One reason for our initial interest in the larval epidermis was that the mosaic pattern found here might be representative of the situation at the blastoderm stage, since morphological studies indicate that the larval epidermis arises from a fairly continuous region of the blastoderm (Poulson, 1950). In the larval mosaics described in this study, the mosaic patterns were quite variable. In some mosaics, the borders were relatively straight; in others, extremely convoluted. However, an extrapolation from any of these patterns, or from their averages, back to the blastoderm stage requires a more quantitative understanding of the developmental events intervening between that stage and final epidermal differentiation. The dorsal-ventral extent of the epiderma1 primordium. After blastoderm formation, the precursor cells for the mesoderm and the endoderm are brought into the interior of the embryo by invaginations at the ventral midline and at the anterior and posterior ends of the embryo (Sonnenblick, 1950; Turner and Mahowald, 1976). In order to estimate the number of cells lost from the surface in the ventral region of the embryo during gastrulation, we have plot-

TABLE 2 OF MOSAICISM FOUND IN DIFFERENT LARVAL AND ADULT STRUCTURES A TOTAL OF 140 LARVAL AND 631 ADULT GYNANDROMORPHS)

(DATA

DERIVED

FROM

Segment

Tl

T2

T3

Al

A2

A3

A4

A5

A6

A7

A8

Mean

Total larval epidermis” Total dorsal histoblast cluster Ventral histoblast cluster Tergites Sternites Legs Genitalia

48.5

63.4

73.9

79.3

82.9

83.6

85.7

83.5

78.1

70.1

-

-

-

-

-

6.9

8.7

8.0

10.9

7.6

8.7

2.2

-

8.8 f 1.3”

-

-

-

7.2

7.2

11.6

6.9

7.6

6.9

4.7

-

8.0 f 2.0”

24.6

21.6

26.3 -

15.3 6.4 -

17.7 8.1 -

15.5 8.3 -

15.6 9.6 -

12.9 5.5 -

27.0

15.4 f 1.7” 7.5 f 1.6 -

_ 25.3 ___-

a Frequencies of mosaicism calculated for entire segment, both right * Average of values from the second through the sixth segments.

and left sides.

-

SZABAD,

SCH~~PBACH,

AND

WIESCHAUS

ted the frequency with which the mosaic border cut through subsections of equal length along the circumference of the larva. The accumulation of borders at the ventral midline represents a surplus of 16% and would arise if the invagination beginning at gastrulation involved 16% of the blastoderm cells. Very similar estimates have been obtained from cell counts on sectioned embryos (17%; Poulson, 1950), from scanning EM studies (20%; Turner and Mahowald, 1977), and from uv laser irradiations (20%; Lohs Schardin et al., 1979). Although the border frequency is highest exactly over the ventral midline defined by the muscle attachment sites, the surplus extends about three cells to its right and left. We also found several larvae where the mosaic border ran in a straight line in the anterior-posterior direction, but significantly to one side of the ventral midline of the precisely defined morphological pattern (Fig. 2d). If the surplus of mosaic borders in the ventral region represents solely the invagination of the mesodermal precursor cells during gastrulation, the spread of six cells indicates that the point where the gastrulation furrow forms need not correspond with (and thus does not directly determine) the line of right-left symmetry in the differentiated larva. This is similar to the results found for the adult, where a developmental restriction between right and left sides arises only late, if ever, during the development of the imaginal discs (Steiner, 1976; Dubendorfer, 1977; Schupbach et al., 1978). The small peak overlying the dorsal midline is probably related to extraembryonic membranes formed in most insects during germ band extension and eliminated as the two lateral sides of the embryo grow dorsally. In Drosophila, these membranes are derived from the dorsal region of the blastoderm between the head fold and the posterior migut invagination. The peak we have observed in this region is small and could be accounted for by the loss of 3-4

Larval

Epidermal

Mosaics

267

blastoderm cells per circumference. It is also possible that the cells of the extraembryonic membranes are not lost but instead are reincorporated into the larval epidermis (Poulson, 1937,195O). If this is the case, the higher border frequency in the dorsal region of the larvae might reflect the lower extent of cell proliferation occurring in the membranes during embryogenesis. Number ofprimordial cells and cellproliferation. Cell divisions are observed to occur very infrequently in the epidermal primordium between 5 and 10 hr of development (Poulson, 1950; Madhavan and Schneiderman, 1977). Final differentiation begins at about 12 hr (Poulson, 1950; S. Eichenberger, personal communication). The 7500 epidermal cells should therefore derive from a fairly large fraction of the cells at the blastoderm stage. In the larval epidermis, circular areas containing 46 cells were mosaic in 23% of the cases, which is similar to the frequency of mosaicism of a single leg in the adult population and indicates that 46 larval epidermal cells are drawn from an approximately equal area of the blastoderm as are the precursor cells for an adult leg. This correspondence takes on additional interest given the observation of Madhavan and Schneiderman (1977) that the leg imaginal discs in the first larval instar contain 36 to 42 cells. That the number of cells in the two structures is similar both at the blastoderm stage and in the first-instar larvae suggests that during embryonic development cell divisions occur with equal frequency among the precursors of the larval epidermis and the imaginal discs. Clonal analysis indicates that about 10 blastoderm cells contribute to each adult leg (Bryant and Schneider-man, 1969; Wieschaus and Gehring, 1976a; Wieschaus, 1978). Judged from their similar frequency of mosaicism, the 46 epidermal cells should also arise from an area of 10 blastoderm cells. The entire larval epidermis of segments Tl-A7 would thus arise from an area

268

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of 1630 cells (7500/46 X 10). In order to make 7500 differentiated epidermal cells, these 1630 cells must divide 2.2 times. A possible criticism of these estimates concerns the number taken for the leg precursors at the blastoderm stage. The figure of 10 cells derives from clonal analysis and may represent an underestimate due to X ray-induced cell death (Haynie and Bryant, 1977) or to the possibility that some cells within the precursor area for each leg do not give rise to scoreable adult structures. Since the leg primordia are roughly circular (Wieschaus and Gehring, 1976b), their maximum size is limited by the width of the individual segments at the blastoderm stage. Irradiations with a uv laser have yielded estimates of three to four cells for the segment width at the blastoderm stage (Lohs-Schardin et al., 1979). Thus an upper limit for the number of cells included in a leg primordia would be about 15. Fifteen cells would raise our estimate of blastoderm precursors for the entire larval epidermis of segments T1-A7 to 2400, and the number of cell divisions would be lowered to 1.6. Taking into account the invagination and loss of cells in the ventral and perhaps dorsal region, the width of the primordium would represent three-fourths of the blastoderm circumference, or about 60 cells. If the primordium consists of 2400 cells and its width is 60 cells, then we can estimate its length in the anterior posterior direction to be 2400/60 or 40 cells. An estimate of 1600 total precursors yields a length estimate of 27 cells. These values are reasonably close to those obtained from the uv laser studies (i.e., 35 cells; Lohs Schardin et al., 1979). The frequencies of mosaicism used to estimate the total number of epidermal precursors were average values, derived from all storable regions of the epidermis. In principle, it should be possible, by comparing the frequencies in different regions, to determine whether different segments arise from different numbers of precursors. In

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FIG. 9. The frequency of mosaicism of roundshaped areas of epidermal cells. The number of epidermal cells in each area (values in parentheses) was calculated by determining the fraction of the total area of a single segment the circular area occupied and assigning an average number of 700 larval and imaginal cells to a segment. The frequencies of mosaicism are plotted against the diameter of the area using cell diameters as the unit of measurement. The linear relationship is expected from the formula originally proposed by Hotta and Benzer (1972), whereby the frequency of mosaicism is proportional to the diameter of the primordium.

our preparations, the lowest frequencies of mosaicism were found in the first two thoracic and eighth abdominal segments (Table 2; see also Fig. 6). Due to the tapering of the larva at the anterior and posterior ends, these segments also have fewer differentiated cells, and it is indeed possible that they arise from a smaller number of precursors. However, these regions were the parts most likely to be damaged when the fiied larva was inverted. It was often difficult to follow the convolutions in the mosaic border in these regions, and thus the values presented in Table 2 and Fig. 6 may be substantial underestimates for these segments. Within the remainder of the larvae, there are no dramatic differences in mosaic border frequency in different segments. In particular, the first and second abdominal segments of the larva have approximately

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the same frequency of mosaicism, even though in the adult, the first abdominal tergite is much more frequently mosaic (Lawrence et al., 1978; see also Table 2). Sturt distances and cell lineage restrictions within the epidermal primordium. Within a given larval segment, the mosaic border cuts through subsections in the anterior-posterior and dorsal-ventral directions with roughly equal frequencies, 4.3 and 4.6% Each subsection is about two differentiated epidermal cells wide and consists, on an average, of two cell interfaces. Thus the probability of a single cell interface being mosaic is 2.2%, and the distance between adjacent epidermal cells, 2.2 sturts. Given the cell divisions occurring during embryogenesis, this figure must be smaller than the sturt distances between adjacent blastoderm cells, since at later stages, the same mosaic border is shared by a larger number of cells. It is possible to show that the probability of a mosaic cell interface decreases by 2+ with each round of cell division, as long as the direction of the division is random, and as long as the proliferation is not accompanied by additional cell mixing (Wiechaus, 1978). Assuming two rounds of cell division during embryogenesis, we can calculate the probability at the blastoderm to be 4.4. When imaginal disc precursors are marked at the blastoderm stage using X ray-induced mitotic recombination, the clones are often very large. However, they are never found to give rise to structures of different segments (Wieschaus and Gehring, 1976a; Steiner 1976; Lawrence and Morata, 1977). This observation suggested the possibility that segmentation arises as a cell lineage restriction early during embryogenesis, perhaps at the blastoderm stage. When, in the present study, mosaic borders were plotted along the anteriorposterior axis (Fig. 6), a series of peaks was found in subsections overlying the segment boundaries. This result is expected if the establishment of segmental restriction in

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the larval epidermis is followed by cell proliferation within each segment. Before segmentation, the mosaic border should fall equally frequently between any two adjacent cells in the primordium. Cell proliferation occurring after segmentation results in a decrease in this probability for cells within a given segment. In contrast, cells which sit on opposite sides of the segment boundary in the differentiated epidermis must always be represented by two separate cells from the time when segments are established. The sturt distance across the segment boundary in the differentiated epidermis must therefore be at least as high as the probability with which the mosaic border fell between adjacent cells at the time when segments were established. In our preparations the mosaic borders were found almost twice as frequently in subsections overlying the segment boundary as elsewhere within the segment (7.6% as compared to 4.3 or 4.6%). Subsections overlying the segment boundary should also consist, on the average, of two cell interfaces, but only one of these would actually be the segmental interface. The other cell interface normally involved in the subsection should be mosaic with the same frequency as the average interface within a segment (2.2). Using this value, we can calculate the value for the segmental interface to be 5.4. This figure is of the same magnitude as that calculated for the probability of the mosaic border falling between adjacent blastoderm cells (4.4), supporting the suggestion that, as with imaginal structures, segmentation in the larval epidermis primordium arises at the blastoderm stage. The higher value measured across the segment boundary in the larva might indicate that adjacent cells in the differentiated epidermis are sometimes formed by cells which are not adjacent at the time when segmentation is established. This could occur if growth in the segments is irregular, if occasional cells are lost at the segment boundary, or if the segments slide against each other during

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germ band elongation or dorsal closure. The absolute values reported here are probably overestimates to a certain extent, and their extrapolation to the actual blastoderm should be made with caution. They are dependent on the assumption that, during germ band extension, no major cell rearrangement occurs within the primordium. Moreover, if the values are corrected for the fact that our larval mosaics probably represent only 84% of the larval gynandromorphs, they fall to 3.7 sturts between adjacent blastoderm cells and 4.5 sturts across the segment border. The larval and the adult abdomens. In Calliphora and in Drosophila the precursors of the abdominal epidermis of the adult have been identified as small, diploid cells clustered in three nests of histoblasts per hemisegment within the sheet of polytenic larval epidermal cells (Bautz, 1971; Pearson, 1972; Madhavan and Schneiderman, 1977; Lawrence et al., 1978). In the present study we found a very close correspondence in genotype between the histoblast cells and the immediately surrounding larval cells. The similarity of the fate maps of larval and adult abdomens is therefore not surprising. From this close correspondence, one can conclude that the arrangement of the imaginal precursors of the abdomen at the blastoderm stage is similar to their arrangement in the differentiated larval epidermis, i.e., that the precursors for the individual histoblast nests are not adjacent to each other but instead are situated among the precursors for the larval epidermis. The distinction between larval and adult fates is therefore a decision which singles out small isolated groups of imaginal cells in what is initially a single sheet of cells. Future cell lineage experiments may reveal whether this segregation has already been made at the blastoderm stage or whether it takes place later during the development of the epidermis. and

We would like to thank Rolf Christiane Nusslein-Volhard

Nothiger, Ilan for helpful

Deak, com-

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ments and criticisms throughout the course of these experiments and Cs. Fajszi for designing the computer program used in calculating sturt distances. This work was supported by grants from the Swiss National Science Foundation to Rolf Ndthiger (Nos. 3.081.73 and 3.741.076) and by a UNDP fellowship to Janos Szabad. REFERENCES BAUTZ, A. M. (1971). Chronologie de la mise en place de l’hypoderme imaginal de l’abdomen de Calliphora erythrocephala Meigen (Insecte, Diptbre, Brachycere). Arch. Zool. Exp. Gen. 112, 157-178. BRYANT, P. J. (1970). Cell lineage relationships in the imaginal wing disc of Drosophila melanogaster. Develop. Biol. 22,389-411. BRYANT, P. J., and SCHNEIDERMAN, H. A. (1969). Cell lineage, growth and determination in the imaginal leg discs of Drosophila melanogaster. Develop. Biol. 20,263-290. D~~BENDORFER, K. (1977). “Die Entwicklung der und weiblichen Genital-Imaginalmannlichen scheibe von Drosophila melanogaster: Eine klonale Analyse,” Ph.D. Thesis. University of Zurich, Schweiz. GARCIA-BELLIDO, A., and MERRIAM, J. R. (1969). Cell lineage of the imaginal discs in Drosophila gynandromorphs. J. Exp. 2001. 170,61-76. GARCIO-BELLIDO, A., and MERRIAM, J. R. (1971a). Parameters of the wing imaginal disc development of Drosophila melanogaster. Develop. Biol. 24,6187. GARCIA-BELLIDO, A., and MERRIAM, J. R. (1971b). Clonal parameters of tergite development in Drosophila. Develop. Biol. 26, 264-276. GUERRA, M., POSTLETHWAIT, J. H., and SCHNEIDERMAN, H. A. (1973). The development of the imaginal abdomen of Drosophila melanogaster. Develop. Biol. 32, 361-372. HALL, J., GELBART, W., and KANKEL, D. (1976). Mosaic systems. In “The Genetics and Biology of Drosophila” (M. Ashburner and E. Novitski, eds.), vol. I, pp. 264-314. Academic Press, London, New York. HAYNIE, J. L., and BRYANT, P. J. (1977). The effects of X-rays on the proliferation dynamics of cells in the imaginal wing disc of Drosophila melanogaster. Wilhelm Roux Arch. 183,85-100. HINTON, C. W. (1955). The behaviour of an unstable ring-chromosome of Drosophila melanogaster. Genetics 40,951-961. HOTTA, Y., and BENZER, S. (1972). Mapping of behaviour in Drosophila mosaics. Nature 240, 527-535. HOTTA, Y., and BENZER, S. (1973). Mapping of behaviour in Drosophila mosaics. In “Genetic Mechanisms of Development” (F. Ruddle, ed.), pp. 129167. Academic Press, New York. JANNING, W. (1972). Aldehydeoxidase as a cell marker for internal organs in Drosophila melanogaster.

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Naturwissenschaften 59,516-617. JANNING, W. (1976). Entwicklungsgenetische Untersuchungen an Gynandern von Drosophila melanoguster. IV. Vergleich der morphogenetischen Anlageplane larvaler und imaginaler Strukturen. Wilhelm Roux Arch. 179.349-372. JANNING, W. (1978). Gynandromorph fate maps in Drosophila. In “Results and Problems in Cell Differentiation: Genetic Mosaics and Cell Differentiation” (W. Gehring, ed.). Springer-Verlag, BerlinHeidelberg-New York. KUHN, D., and CUNNINGHAM, G. (1978). Aldehydoxidase distribution in Drosophila melanogaster mature imaginal discs, histoblasts and rings of imaginal cells. J. Exp. Zool. 204, l-10. LAWRENCE, P. A., GREEN, S. M., and JOHNSTON, P. (1978). Compartmentalization and growth of the Drosophila abdomen. J. Embryol. Exp. Morphol. 43.233-245. LAWRENCE, P. A., and MORATA, G. (1977). The early development of mesothoracic compartments in Drosophila An analysis of cell lineage and fate mapping and an assessment of methods. Deuelop. Biol. 56, 40-51. LEWIS, E. B. (1978). A gene complex controlling segmentation in Drosophila. Nature 276,565-570. LINDSLEY, D. L., and GRELL, E. H. (1968). Genetic variations of Drosophila melanogaster. Carnegie Inst. Wash. Publ. 627. LOHS-SCHARDIN, M., CREMER, C., and N~RLEIN-VOLHARD, C. (1979). A fate map of the larval epidermis of Drosophila melanogaster: Localized cuticle defects following irradiation of the blastoderm with an ultraviolet laser microbeam. Develop. Biol. 73,239255. MADHAVAN, M. M., and SCHNEIDERMAN, H. A. (1977). Histological analysis of the dynamics of growth of imaginal discs and histoblast nests during the larval development of Drosophila melanogaster. Wilhelm Roux Arch. 183,269-305. NISSANI, M. (1975). Cell lineage analysis of kynurenine producing organs in Drosophila mehnogaster. Genet Res. 26,63-72. PARKS, H. (1936). Cleavage patterns in Drosophila and mosaic formation. Ann. Entomol. Sot. Amer. 29, 350-392. PEARSON, M. J. (1972). Imaginal discs and the abdominal histoblasts of Calliphora erythrocephala (Diptera). Nature (London) 238, 5363, 349-351. POSTLETHWAIT, J. H., and SCHNEIDERMAN, H. A.

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(1971). A clonal analysis of development in Drosophila melanogaster: Morphogenesis, determination and growth in the wild-type antenna. Develop. Biol. 24,477-519. POULSON, D. F. (1937). The embryonic development of Drosophila metanogaster. Exposes Genet. 498111, l-48. POULSON, D. F. (1950). Histogenesis, organogenesis and differentiation in the embryo of Drosophila melanogaster, Meigen. In “Biology of Drosophila” (M. Demerec, ed.), pp. 168-274. Wiley, New York. ROBERTSON, C. W. (1936). The metamorphosis of Drosophila melanogaster including an accurately timed account of the principal morphological changes. J. Morphol. 58,351-400. ROSELAND, C., and SCHNEIDERMAN, H. (1979). Regulation and metamorphosis of the abdominal histoblasts of Drosophila melanogaster. Wilhelm Roux Arch. 186, 235-265. SCH~~PBACH, T., WIESCHAUS, E., and NBTHIGER, R. (1978). The embryonic organization of the genital disc studied in genetic mosaics of Drosophila melanogaster. Wilhelm Roux Arch. 185,249-270. SONNENBLICK, B. P. (1950). “The Early Embryology of Drosophila melanogaster” (M. Demerec, ed.), pp. 62-167, Wiley, New York. STEINER, E. (1976). Establishment of compartments in the developing leg imaginal discs of Drosophila melanogaster. Wilhelm Roux Arch. 180, 9-30. SZABAD, J. (1978). Quick preparation of Drosophila for microscopic analysis. DZS 53, 215. TURNER, R. F., and MAHOWALD, A. P. (1976). Scanning electron microscopy of Drosophila embryogenesis. 1. The structure of the egg envelope and the formation of the cellular blastoderm. Develop. Biol. 50.95 108. WIESCHAUS, E. (1978). Cell lineage relationships in the early Drosophila embryo. In “Results and Problems in Cell Differentiation. Genetic Mosaics and Cell Differentiation” (W. Gehring, ed.). SpringerVerlag, Berlin-Heidelberg-New York. WIESCHAUS, E., and GEHRING, W. (1976a). Clonal analysis of primordial disc cells in the early embryo of Drosophila melanogaster. Develop. Biol. 50, 249-263. WIESCHAUS, E., and GEHRING, W. (1976b). Gynandromorph analysis of the thoracic disc primordia in Drosophila melanogaster. Wilhelm Roux Arch. 180.31-46.