DEVELOPMENTAL

57, 403-416 (1977)

BIOLOGY

Scanning

Electron

Microscopy of Drosophila Em bryogenesis

II. Gastrulation F. RUDOLF Department Received

TURNER

of Zoology, October

18,1976;

and Segmentation AND

Zndiana

melanogaster

ANTHONY

University,

accepted

P. MAHOWALD

Bloomington,

in revised

form

Indiana February

47401 8,1977

The sequence of gastrulation events in Drosophila melanogaster, starting with the cellular blastoderm and culminating in a segmented embryo, have been studied with scanning electron microscopy (SEM). Extensive use is made of dissected embryos to illustrate changes taking place within the embryo during gastrulation. During the first 15 min of gastrulation, the mesodermal portion of the germ band is established by the invagination of approximately 1000 cells through the ventral furrow. The primordia for the proctodeum and hindgut are shown to form during early gastrulation. Detailed examination of the surfaces of invaginating primordia shows similarities to other systems and suggests possible underlying mechanisms. Germ band elongation and the formation of the amnioserosa are described. At the time of segmentation, three pairs of rudimentary cephalic appendages develop posterior to the cephalic furrow. Tracheal pits invaginate on all eight abdominal segments and on the second and third thoracic segments. Modifications of the embryonic fate map are discussed. INTRODUCTION

The most dramatic changes in the structure of an embryo occur during the rapid cellular movements which transform the simple blastula or blastoderm stage into the three germ layers of the gastrula. Some aspects of these developmental changes have been shown to depend upon the developmental program established in the oocyte by the maternal genome (Sturtevant, 1923; Briggs, 1973; Rice, 1973; Rice and Garen, 1975; Zalokar et al., 1975). In addition, a number of recessive embryonic lethal mutations appear to affect preimplantation stages in mice (Bennett, 1964) and the earliest stages of gastrulation (Ede, 1956a, b, c) in Drosophila. More detailed analysis of the increasingly rich supply of mutations in Drosophila melanoguster (Wright, 1970; King, 1970; King and Mohler, 1975) prompts the reinvestigation of much of the descriptive embryology of early development, utilizing newer procedures such as transmission (TEM) and

scanning electron microscopy (SEMI. SEM provides abundant opportunities for visualizing the three-dimensional changes occurring during gastrulation (e.g., Monroy et al., 19761. Because of the complexity of the changes, TEM analysis would be very difficult. In this paper, we will describe the processes of gastrulation in wild-type embryos using SEM, from the time of the first cellular movements to the completion of germ band extension and the appearance of segmentation. Additional papers in this series will complete the description of the wild-type embryo and begin an analysis of selected mutations. MATERIALS

AND

METHODS

Embryos from a Drosophila melanogasOregon R stock were used in these studies. The embryos were prepared for SEM by procedures used previously (Turner and Mahowald, 1976). Briefly, chorions of embryos of the appropriate age were removed, and the embryos were fixed in a trialdehyde mixture (Kalt and Tanter

403 Copyright 0 1977 by Academic Press, Inc. All rights

of reproduction

in any form reserved.

ISSN

0012-1606

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DEVELOPMENTAL

BIOLOGY

dler, 1971) following permeabilization in heptane (Zalokar, 1971). After removal of the vitelline membrane, the embryos were treated with 1% osmium tetroxide, followed by ethanol dehydration. To reveal internal details of the embryo, selected stages were dissected in the appropriate plane with tungsten needles while in the trialdehyde fixative. Some of the dissected embryos were sonicated in an ultrasonic cleaner (L and R, Model 3206) to remove loose yolk prior to osmication. Following critical-point drying, the embryos were mounted on stubs with Epoxy glue, coated with carbon and gold-palladium, and examined with an ETEC Autoscan SEM operated at 20 kV. RESULTS

(I 1 Synopsis Gastrulation consists of an integrated series of complex movements during which the primary germ layers are formed and the embryonic axis or germ band is elaborated. Detailed histological descriptions of these movements in Drosophila have been provided by Sonnenblick (1950) and Poulson (1950). A brief synopsis of these changes will assist in following the detailed analysis. Following the completion of the cellular blastoderm at 3.5 hr (Turner and Mahowald, 1976), the presumptive mesoderm invaginates by means of a long midventral furrow. The endoderm forms by means of a series of invaginations. A small pocket of cells along the anterior ventral region of the embryo invaginates at the same time as the mesoderm and separates from the surface forming the anterior midgut rudiment. Subsequently, at 4.5 hr, a new invagination forms in the same region producing the large stomodeal pocket to which the anterior midgut rudiment becomes attached. Posterior to the ventral furrow, two further endodermal invaginations occur at 3.75 hr: the first, as a pouch-like invagination below the pole cells, forms the posterior midgut rudiment; and the second, as a fold of mid-

ventral cells between the pole cells and the ventral furrow, forms the prospective proctodeum and hind gut. From 3.75 to 5 hr, the embryonic germ band forms by the extension of the ventral mesoderm and the overlying ectoderm around the posterior tip to a position adjacent to the future head region. This germ band extension moves the future proctodeum adjacent to the head segments along the dorsal surface of the embryo. Following completion of the full extension of the germ band, segmentation and histogenesis occur. (2) Formation

of the Ventral

Furrow

The first evidence of the invagination of the presumptive mesoderm tissue along the midventral surface is a change in the surface morphology of the midventral blastoderm. At the completion of blastoderm formation, the free surface of the cells becomes relatively smooth (Turner and Mahowald, 1976). Then, along the midventral surface of the embryo, bulbous processes appear, rapidly obliterating the outlines of individual cells (Figs. 1 and 2). Within a few minutes, a long fold appears along the ventral surface (Fig. 3). The length of the initial furrow varies. In some embryos, the furrow stretches along 50% of the ventral surface (Fig. 3), reaching anteriorly only to the cephalic furrow (discussed in the next section). In other instances, the furrow extends for approximately 60% of the embryo’s length, reaching anteriorly to the future anterior midgut invagination (Fig. 1). The changes in cellular shape occurring during ventral furrow formation can be seen clearly in cross-fractured embryos. Initially, the midventral cells change from tall columnar to wedge-shaped cells the apices of which have become constricted at the forming furrow and the bases have become enlarged (Fig. 4). At this time, the furrow produces a broad indentation in the central yolk sac. Subsequently, the cells shorten (Fig. 5), and the furrow deepens and closes to produce a cylinder of cells

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attached to the surface at the fused lips of the furrow. The surface cells adjacent to the ventral furrow form a thin elongate layer which stretches from the yolk sac membrane, around the invaginated mesoderm, to the midline (Fig. 5). At the edge of the forming furrow, the apices of the cells appear to be stretched toward the furrow (Figs. 2 and 4), whereas the bases of the cells involved appear relatively immobile. Rick011 (1976) has found many thin peglike cytoplasmic bridges between the blastoderm cells and the syncytial yolk sac membrane. We have confirmed his observations concerning the dorsal and lateral cells (Fig. 4), but we have not found any bridges between the invaginating midventral cells and the yolk sac. Since TEM analysis of thin sections indicates that some are present (Rickoll, personal communication), the failure to detect them with SEM in dissected embryos is possibly due to their infrequent occurrence. The presence of these cytoplasmic bridges to the yolk sac may provide a stable base for the lateral cells during the active surface movements along the midventral surface. It is possible to give an estimate of the numbers of cells which invaginate to form the mesoderm. The furrow initially forms along 50-55% of the ventral surface and becomes extended to 60-65% of the embryo’s length. Since some of the posterior lengthening is due to the drawing of ventral cells around the posterior tip as the posterior midgut invagination moves anteriorly (see below), the actual percentage of the length of the ventral surface that invaginates is probably no more than 60%. Based on counts of cells within the ventral furrow in embryos broken in half (e.g., Fig. 5), a band of cells approximately 20 cells wide invaginates. Counts of cells along the length of the total blastoderm indicate that there are 80-85 cells in this plane. Since the ventral furrow extends for 60% of the ventral surface, approximately 1000 cells invaginate. Previously, we have

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determined that the total number of cells forming the blastoderm varies between approximately 5600 and 6500 cells (Turner and Mahowald, 1976; Zalokar and Erk, 1976). Thus, between 15 and 18% of the blastoderm layer invaginates within the first 15 min of gastrulation to form the mesodermal portion of the germ band. (3) Formation

of the Cephalic Furrow

Simultaneously with the formation of the ventral furrow, an additional invagination of cells appears as a transverse fold across the lateral surface of the embryo, one third of the distance from the anterior tip (Figs. 1 and 3). There are no prominent bulbous projections at the free surfaces of cells at the site of this invagination (Fig. 7). The invagination appears to start because a transverse row of lateral cells shorten while remaining attached to the yolk sac membrane (Fig. 6), drawing the apical portions of adjacent cells toward the yolk. Initially, these cells fold in along the lateral surfaces of the embryo (Fig. l), and, subsequently, the fold expands across the ventral and dorsal surfaces. The invagination also rapidly deepens as it expands so that, in a few minutes, a furrow has formed which reaches through the thickness of the original blastoderm layer (Fig. 8). The cells of the cephalic furrow remain attached to the ectoderm as a continuous cellular layer, instead of separating as an internal cellular mass as occurs during formation of the mesoderm. At the completion of germ band elongation, the cephalic furrow disappears without leaving a trace of itself in any larval structure. (4) Anterior

Midgut

Znvagination

The ventral furrow becomes extended 50 pm past the cephalic furrow at which point the invagination widens into a Tshaped depression (Fig. 27) which becomes the anterior midgut rudiment. The surface of the invaginating cells shows the same bulbous morphology which characterizes the ventral furrow (Fig. 28). The anterior

TURNER AND MAHOWALD

SEM

midgut rudiment stays patent to the surface for a considerably longer time than the adjacent portion of the ventral furrow but, by 4-4.5 hr, the surface layer closes over this invagination. During the formation and closing of the anterior midgut rudiment, distinctive long microvilli appear on the surface of the embryo in this region (Fig. 29). Similar cellular processes are also evident at the inception of the stomodeal invagination which forms anterior to the closed anterior midgut (Ede and Counce, 1956). (5) Posterior Midgut and Proctodeal Invaginations A fourth invagination begins at the posterior tip. During formation of the cellular blastoderm, the pole cells are embedded within the surface of the blastoderm and are frequently difficult to distinguish from the blastoderm cells in a SEM surface view (Turner and Mathowald, 1976). As gastrulation begins, the surfaces of the blastoderm cells become relatively smooth, and the pole cells now protrude as a plaque of

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cells on the dorsal surface of the posterior tip (Figs. 1 and 3). The surface of the blastoderm cells below and in the immediate vicinity of the cluster of pole cells develops the same bulbous features (Fig. 20) which characterize the cells of the early ventral furrow. This disk-shaped region (posterior midgut rudiment) first flattens (Fig. 19) and begins to move cephalad along the mid-dorsal surface of the embryo. The anterior border of this region then becomes depressed (Fig. 17) to form a pocket within which the pole cells are located. This pocket or posterior midgut invagination rapidly deepens (Figs. 21 and 24) as it continues to move cephalad along the dorsal surface (Figs. 9-13). The pole cells become internalized within the first 30 min of gastrulation. Subsequent changes of the posterior midgut rudiment will be described along with the elongation of the germ band. A fifth invagination appears after the closing of the midventral furrow and the start of the posterior midgut invagination. The free surface of the blastoderm cells

FIG. 1. Ventral view of a very early gastrula stage embryo. The ventral furrow (V) has started to form along the midventral surface, and the pole cells (p) have begun their anterior-dorsal movement. Note the cephalic furrow (arrow) forming toward the anterior pole of the embryo. 270 x The embryos in Figs. 1,2,19, and 21 are mounted with their posterior tips toward the top of the page. In Figs. 9-16, the embryos are pictured with their anterior tips upward. FIG. 2. A cross-fracture through the region of the ventral furrow shortly after its first appearance. Characteristic bulbous projections (arrow) are seen on the surfaces of the cells which will soon be invaginated. Note the changes in shape of the peripheral portions of blastoderm cells immediately adjacent to the furrow. 1870 X. FIG. 3. Ventral view of an embryo slightly older than the one shown in Fig. 1. The ventral furrow (V) has begun to invaginate: (p) pole cells; (C) cephalic furrow. 245 X. FIG. 4. Transverse break through an embryo at about the stage shown in Fig. 1. The ventral furrow (V) has just started to invaginate. Compare the shape of the cells on either side of the ventral furrow with the remaining columnar blastoderm cells surrounding the yolk sac (Yl. 640 x The peg-like projections which connect the blastoderm cells (B) to the yolk sac membrane (ym) can be seen at higher magnification in the inset. 3800 X. FIG. 5. Transverse break through a ventral furrow at the completion of invagination of the prospective mesoderra. The blastoderm cells overlying the mesoderm have become greatly altered in shape. 1150 x . FIG. 6. Fracture through a very early stage in cephalic furrow formation showing the shortening of the initial cell of the furrow (arrow). 1100 x. FIG. ‘7. Surface view of an early stage of cephalic furrow formation. Compare the relatively smooth surface of the invaginating cells with the bulbous surfaced cells of the ventral furrow in Fig. 2. 1900 x FIG. 8. Sagittal break through the anterior end of an early gastrula stage (ventral side up) showing a later stage in the development of the cephalic furrow (C). Yolk, (Y). 580 x

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DEVELOPMENTAL BIOLOGY

between the posterior end of the closed ventral furrow and the posterior midgut rudiment develops bulbous projections (Fig. 201 similar to those formed at the inception of the ventral furrow and anterior and posterior midgut. A deep central fold quickly forms (Fig. 17) and closes (Fig. 9). Although this newly formed furrow appears to be an extension of the ventral furrow, the two are distinct. In contrast to the ventral furrow, these infolded cells retain continuity with the surface ectoderm as well as with the posterior midgut invagination. The subsequent fate of these cells will be described along with germ band extension. (6) Extension

of the Germ Band

(a) Formation of the mesoderm. Following these five early invaginations which occur during the first 30 min after formation of the cellular blastoderm, most of the subsequent movements of gastrulation in-

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volve the rapidly expanding germ band, consisting of the mesoderm and the overlying ectoderm. Initially, the mesoderm forms a solid rod of cells along the midventral surface (Figs. 21 and 25). The region where the invaginated mesoderm comes into contact with the ectoderm is marked by a high concentration of interdigitating cell processes (Fig. 23). The germ band rapidly lengthens posteriorly, and, as the posterior midgut invagination advances cephalad in front of it (Fig. 24), the germ band stretches around the posterior tip and advances to the opening of the proctodeum (see below). The mesodermal portion of the germ band remains a solid cylinder along the ventral surface during the initial period of elongation (Fig. 25). At the time when the germ band has advanced about 50% of the distance anteriorly along the dorsal surface, the mesoderm begins to spread laterally along the inner surface of the ectoderm (Fig. 30).

FIG. 9. Dorsal view of an embryo after ventral furrow closure. The posterior midgut pocket is beginning to carry the pole cells (p) into the interior of the embryo at the same time that it and the proctodeal invagination (Pr) are being moved anteriorly by the elongating germ band. The posterior transverse fold (PF) is very pronounced at this stage. Five patches of rounded cells are identifiable anterior to the cephalic cleft (0. 200 x. FIG. 10-12. Three embryos illustrating the internalization of the pole cells and continued elongation of the germ band. As the proctodeal invagination moves progressively anteriorly, there is a simultaneous stretching and thinning of a large group of cells peripheral to the germ band to form the amnioserosa (AS). The anterior transverse fold (AF) is lost during this period, and the posterior transverse fold (PF) disappears before the completion of germ band elongation (Fig. 13). The mesectoderm (me) is seen as a double row of cells along the midline of the germ band in Fig. 11. (C) cephalic furrow. Fig. 10: 230 x . Fig. 11: 220 X Fig. 12: 240 x. FIG. 13. Lateral view of an embryo at the completion of germ band elongation. Early evidence of segmentation (arrow) is seen adjacent to the amnioserosa (AS). 230 x . FIG. 14. Lateral view of a 6.5-hr embryo showing an early stage in the formation of the cephalic appendages. The maxillary (Mx) and labial (L) rudiments are distinct, whereas the mandibular (Ma) rudiment is just discernible posterior to the cephalic furrow. Tracheal pits (arrow) and evidence of segmentation are seen along the ventral germ band. 225 x . FIG. 15. Ventral view of an embryo showing a later stage in segmentation. (S) stomodeum. 290 X. FIG. 16. Ventral view of a 7-hr segmented embryo. Tracheal pits (arrows) are visible on the anterior border of the segments T2, T3, Al, and A2. The salivary gland placode (sg) is forming on the labial appendage. 200 X. FIG. 17. Dorsal surface of the posterior end of an early gastrula stage showing the deeply invaginated posterior furrow (arrow) which will become the proctodeal invagination. The pole cells (p) are clustered on top of the posterior midgut rudiment which is beginning to form a pocket. 450 x . FIG. 18. Lateral view of an embryo near tbe completion of segmentation showing the mandibular (Ma), maxillary (Mx) and labial (L) cephalic appendages, three thoracic segments (Tl-T3), and eight abdominal segments (Al-A@. The tracheal pits (arrow) are located near the anterior edge of the last 10 segments. 285 x

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DEVELOPMENTAL BIOLOGY

(b) Dorsal folds and extraembryonic membranes. Two prominent transverse folds appear across the dorsal surface of the embryo shortly after the start of gastrulation. The first and major fold forms during the initial gastrulation movements (Fig. 19), but, quickly, a second minor fold becomes established anterior to the major fold (Figs. 9 and 10). As the germ band extends along the dorsal surface (Figs. lo131, a U-shaped layer of cells develops which stretches from the lateral sides of the germ band around its anterior-most extension. These cells become extremely thin as they become transformed into the amnioserosal layer. As this layer forms, both folds remain (Fig. 11). However, as the germ band advances anteriorly, first the anterior fold disappears (Fig. 12), and then the posterior fold disappears (Fig. 13). The amnioserosa is composed of one continuous sheet of cells joining the cells

VOLUME 57, 1977

at the cephalic furrow with the lateral edges of the ectoderm of the posterior portion of the embryo (Figs. 11-13). The cells of the amnioserosal layer are distinctive. Their large nuclei protrude as bulges within the otherwise flattened cells (Fig. 32). Many short, thin microvilli are present along borders between adjacent cells (not illustrated). The transition in morphology between these cells and the ectoderm is abrupt. The amnioserosal layer frequently lifts off from the underlying acellular yolk sac membrane, suggesting the absence of special attachments (Fig. 32). (cl Formation of the proctodeal invagination. Poulson (1950) has described the proctodeal invagination as forming at the future posterior end of the embryo, between the fifth and sixth hour of development, as a slit-like invagination of large columnar cells. This new invagination is readily vis-

FIG. 19. Dorsal view of an embryo slightly older than that shown in Fig. 3. The posterior midgut rudiment (P) supporting the pole cells is beginning to invaginate as it moves anteriorly. (PF) posterior transverse fold. 230 x FIG. 20. The posterior tip of an early gastrula stage which was sonicated after fixation. The pole cells have been dislodged revealing the underlying posterior midgut rudiment with its bulbous surface. Some pole cells were deeply embedded in the blastoderm layer (circle). Posterior to the posterior midgut rudiment, a region of bulbous surfaced blastoderm marks the site which will soon become the posterior furrow (arrow7. 755 x. FIG. 21. Midsagittal fracture through an embryo at a stage similar to that shown in Fig. 10. The pole cells have been lost during sonication. Note the rodlike mesoderm (M) along the ventral surface. (P) posterior midgut; (C) cephalic furrow; (PF) posterior transverse fold; (S) stomodeum. 240 x FIG. 22. Higher magnification of the posterior end of the embryo shown in Fig. 21. The smooth cell surfaces of the wall of the proctodeal slit (Pr) indicate that the cells of the two sides of the embryo do not interdigitate in this region. Contrast this surface with a fractured area of the ectoderm in the germ band region (arrow). (M) mesoderm. 635 x. FIG. 23. A high-magnification view of a portion of the germ band from Fig. 21, showing the proliferations of cell membrane processes at the interface between the recently invaginated mesoderm (M) and the overlying ectoderm (E). 1485 x. FIG. 24. The region of the posterior midgut and the enclosed pole cells (p) in a broken embryo somewhat older than the one shown in Fig. 21. The anterior dorsal edge of the posterior midgut is continuous with the edge of the proctodeal slit (Pr). The gaps seen between cells of the posterior midgut wall (arrow) are a common feature at this stage. Mesodermal cells (M) are evident at the anterior edge of the germ band. 530 X FIG. 25. An oblique transverse break through an embryo at a stage slightly older than that shown in Fig. 11, looking toward the posterior end. The fracture plane has passed through the rod-shaped mesoderm (M) on the ventral side and through the posterior midgut (P) on the dorsal side. The two halves of the embryo along the mid-dorsal line are appressed in this region (arrow). A small portion of the amnioserosa (AS) in an early stage of formation can be seen lateral to the posterior midgut. (p) pole cells. 530 x . FIG. 26. Portion of an embryo (stage approximately that of Fig. 18) cross-fractured to show the proctodeal slit terminating at the proctodeal opening (arrow). The maxillary appendages (Mx) can be seen on either side of the stomodeum (S). (AS) amnioserosa. 440 x .

.

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DEVELOPMENTAL BIOLOGY

ible in SEM views of embryos at this time (Fig. 26). A medial slit appears at 6 hr within the columnar cells, which form the dorsal surface of the posterior midgut invagination at the opening into the space covered by the aminoserosa (Fig. 24). At 8 hr, the slit has become continuous with a small circular opening (Fig. 26) which will develop into the true proctodeal or anal opening following contraction of the germ band (unpublished observation). The proctodeal slit will subsequently close along the midline of the anal segment. Although the proctodeal invagination becomes apparent as an opening at 6 hr, the cells which form the proctodeum are visible from the time of the formation of the ventral fold posterior to the germ band (Fig. 17). Dorsal views of the posterior surfaces of embryos during germ band elongation (Figs. 9 and 10) show a central line during invagination of the posterior midgut. Sagittal breaks along the midline (Figs. 22 and 24) show a clean separation of cells in this region, and cross-fractures (Fig. 25) suggest that there is no cellular interdigitation along the center line. After these cells have invaginated behind the presumptive posterior midgut, the proctodeal cells separate, producing the slit as described above. Thus, the future proctodeaum clearly originates from the cells along the midventral line posterior to the ventral furrow. (7) Segmentation As stomodeal invagination is occurring on the anterior ventral surface, segmentation becomes apparent externally in the region posterior to the cephalic furrow. The first indication of segmentation occurs adjacent to the two rows of thin mesectoderm cells (Fig. 15), where periodic clusters of protruding surface cells are located. These cells may be the neuroblasts. Similarly, on the lateral edge of the ectoderm adjacent to the amnioserosa, deep invaginations of the ectoderm indicate the location of future segments (Fig. 13). Inter-

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nally, the mesoderm becomes clearly divided into segmented masses (Poulson, 1950). Three rudimentary cephalic appendages form which have not been noted in Drosophila embryos previously, but have been described in Calliphora (Schoeller, 1964) and other insects (Anderson, 1973). The most anterior pair of appendages (mandibular) forms laterally, just posterior to the cephalic furrow (Fig. 14), as a small triangular structure. Posterior to this structure, two pairs of prominent appendages form (the maxillary and the labial) on the ventral surface (Figs. 14 and 18). The deepening of the stomodeum (Fig. 16) draws the surface of the embryo posterior to this invagination anteriorly, so that the cephalic appendages are drawn toward the midline and anteriorly toward the stomodeum. The cephalic furrow is no longer evident as the process of head involution begins (Fig. 18). On each labial appendage, a circular depression, the salivary gland placode, appears on the ventral surface (Fig. 16). The remaining five segments along the ventral surface are the three thoracic (T) and first two abdominal (A) segments (Fig. 18). Segment A3 is at the posterior tip and A4-A8 stretch anteriorly along the dorsal side of the embryo. Shortly after the appearance of the segments, paired trachael pits form on the anterior lateral surface of 10 segments: T2, T3, and Al through A8 (Figs. 14, 16 and 18). Previous descriptions of dipteran development (Poulson, 1950; Anderson, 1973) have indicated that tracheal invaginations are also found on Tl, but we have found no evidence of this invagination with SEM. As the surface segmental boundaries become deeper, the pits are drawn into the grooves (Fig. 18). Each of the tracheal pits consists of an invagination of the ectoderm into the mesoderm layer where the pit forms a T-shaped base. During shortening of the germ band, the T-shaped bases fuse to form tracheal trunks, and the original surface connections disappear (Poulson,

FIG. 27. Ventral surface of the anterior tip of an early gastrula stage embryo, illustrating an early stage in the formation of the anterior midgut rudiment at the anterior end of the ventral furrow (V). 580 x . FIG. 28. A higher-magnification view of the anterior midgut rudiment showing the characteristic bulbous projections on the surfaces of the invaginating cells. Compare with the invaginating ventral furrow (Fig. 2) and the posterior midgut rudiment (Fig. 20). 2200 X. FIG. 29. A small portion of the area of the posterior midgut rudiment after its invagination, showing the long filamentous projections on the cells which cover this region. 3600 x . FIG. 30. A portion of a germ band from a broken sonicated embryo. The mesoderm (M) is spreading over the inner surface of the ectoderm (E). Note the advancing edge of the mesoderm (arrow). (n) neuroblast. 700 X. FIG. 31. Fracture near the anterior tip of an embryo at a stage similar to that shown in Fig. 9, showing the morphological differences between the rounded cells and the adjacent columnar blastoderm cells (B). The basal portions of the columnar cells rest on the yolk sac membrane (ym). 1440 x . FIG. 32. Cross-fracture through a small area of amnioserosa and underlying yolk sac. Bulges in the amnioserosal cells represent the positions of nuclei. (ym) yolk sac membrane. 1980 x 413

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1950). The posterior spiracle develops secondarily in the lateral portion of the eighth abdominal segment (unpublished results). (8) Neuroblasts There is some indication of the segmental location of presumptive neuroblasts along the ventral surface of the embryo at 4.5 hr (Fig. 15). Poulson (1950) has described an increase in size of the neuroblasts at the time when they withdraw from the surface and begin a series of characteristic unequal cell divisions in the region between the ectoderm and mesoderm. Large cells are frequently found embedded between these two layers (Fig. 30). Along the midline, two rows of thin mesectoderm cells (Fig. 11) stretch from the ectoderm layer to the mesoderm, separating the neuroblasts on each side of the embryo. These mesectoderm cells retain their connection to the mesoderm during its subsequent differentiation (Poulson, 1950). A dorsal band of tightly packed cells stretches anteriorly from the cephalic furrow to a point 50 pm from the anterior tip, where it splits into two thin bands of cells projecting laterad (Figs. 9 and 10). Approximately 150 pm from the anterior tip, two additional lateral bands of small cells connect to the dorsal band. In cross-fractured embryos, the bands are seen to be formed of tall columnar cells, whereas, between the lateral bands, on each side are clusters of large cuboidal cells (Fig. 31). Another cluster of cuboidal cells is located anterior to the central band of columnar cells. The change in shape from columnar blastoderm cells to cuboidal surface cells occurs rapidly. For the first 15 min after the start of gastrulation, the cells of the anterior dorsal region are columnar. Then, at the time the prospective proctodeal fold closes, the patches of large surface cells appear, separated by thin columnar cells. In Feulgen-stained whole mounts, clusters of mitotic cells can be found in the regions where the round surface cells are located.

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These cells are probably not true neuroblasts because their plane of division is parallel to the surface, whereas neuroblasts always divide perpendicular to the surface (Poulson, 1950; personal communication). However, it is possible that these cells are precursors of the true neuroblasts. The initial appearance of five clusters may be a relic of the primitive segmental nature of the insect brain (cf. Anderson, 1973). DISCUSSION

The most remarkable feature of gastrulation is the rapidity of the cellular movements which produce the three primary germ layers. The mesoderm forms from approximately 1000 cells which invaginate along the ventral surface during the first 15 min. Then, within 30 min, three separate invaginations produce the major rudiments of the endoderm: the anterior midgut which forms as a small invagination at the anterior end of the ventral furrow; the proctodeal fold which forms posterior to the mesoderm; and finally, the posterior midgut rudiment which forms beneath the pole cells as a large pocket within which the pole cells are included. The only additional invagination occurs 1 hr later when a new infolding occurs in front of the closed anterior midgut invagination. This stomodeal pocket quickly enlarges, grows posteriorly, and becomes joined to the previously invaginated anterior midgut rudiment. The forces involved in these rapid movements are not clear. SEM of dissected embryos suggests two events for the formation of the ventral furrow: first, the appearance of many bulbous projections on the invaginating surfaces, suggesting the presence of a cytoplasmic contractile system similar to that found associated with cell furrows (Schroeder, 1973); second, a change in the shape of the midventral cells from a simple cuboidal to a pyrimidal shape. Thus, the invagination could result from the change in cell study caused by a

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cytoplasmic contractile system acting at the surface of the midventral cells; subsequently, this continued contraction would draw together the external surfaces of these cells, producing the rod-like mesodermal mass. Similar hypotheses pertaining to neurulation in other animal systems have been reviewed by Karfunkel (1974). The cephalic furrow appears to form by a different mechanism. There is no evidence of bulbous projections on the surface. The furrow appears to begin because a single oblique row of cells contracts toward the yolk. Although we have not found bridges between these cells and the syncytial yolk sac in fractured embryos, some type of stable junction must be present so that the cells contract toward the yolk. The adjoining cells must be tightly joined to this row of cells so that a fold or cleft forms. Forces causing the subsequent enlargement of the cephalic furrow are more difficult to envision. The furrow becomes 50-75 pm deep and nearly separates the embryo into two portions. Since large numbers of blastoderm cells are invaginating at the same time through the ventral furrow, the cause is probably not a crowding at the surface. On the other hand, patches of head blastoderm cells are rounding up at this time and dividing at the surface. This process could conceivably result in the spreading of other anterior surface cells posteriorly where they become part of the cephalic furrow. Since the furrow is a simple fold and never produces a specific embryonic structure (Poulson, 19501, its function may simply be for the temporary “storage” of anterior surface cells until a later time in development. Larval fate maps have been drawn for the Drosophila blastoderm stage from a histological analysis of development (Poulson, 1950), and adult fate maps have been drawn from the analysis of gynandromorph boundaries (Garcia-Bellido and Merriam, 1969; Hotta and Benzer, 1973). From the current study, a few additions to previously published fate maps are possi-

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ble. We have reason to locate the presumptive proctodeal and hindgut regions in the posterior ventral area of the blastoderm. The sequence of cellular changes in the formation of the amnioserosal membrane suggests that the precursor cells of this thin membrane derive from both the middorsal cells and along the deep dorsal folds. Finally, the cephalic appendages form posterior to the cephalic furrow, indicating that this invagination does not separate the larval head and thorax, as previously suggested (Poulson, 1950). Possibly, the most accurate way of producing such fate maps will be by transplanting genetically labeled cells from one embryo to the same site on a differently labeled embryo of the same age and then determining the fate of these cells. Preliminary experiments by Illmensee (personal communication) have indicated that this is possible. This knowledge will assist in determining unequivocally the fate of the cells during gastrulation. This work was supported by a grant from the NIH, No. HD07983. We wish to thank Dr. Donald F. Poulson for helpful discussions and for the opportunity to examine some of his histological preparations of Drosophila embryos. This is publication No. 1048 from the Department of Zoology. REFERENCES ANDERSON, D. T. (1973). “Embryology and Phylogeny in Annelids and Arthropods,” p. 495. Pergamon Press, Oxford. BENNETT, D. (1964). Abnormalities associated with a chromosome region in the mouse. Science 144, 263-267. BRIGGS, R. (1973). Developmental genetics of the axolotl. In “Genetic Mechanisms of Development” (F. H. Ruddle, ed.), pp. 169-199. Academic Press, New York. EDE, D. A. (1956a). Studies on the effects of some genetic lethal factors on the embryonic development of Drosophila melanogaster. Preliminary survey of some sex-linked lethal stocks, and an analysis of the mutant Lffll.Wilhelm Roux Arch. 148, 416-436. EDE, D. A. (1956bl. Studies on the effects of some genetic lethal factors on the embryonic development of Drosophila melanogaster. II. An analysis of the mutant X2. Wilhelm Roux Arch. 148, 437451. EDE, D. A. (1956c). Studies on the effects of some

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