Development 112, 365-370 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

365

Drosophila gastrulation: analysis of cell shape changes in living embryos

by three-dimensional fluorescence microscopy

ZVI KAM2, JONATHAN S. MINDEN1*, DAVID A. AGARD1, JOHN W. SEDAT1 and MARIA

LEPTIN3i

^Department of Biochemistry and Biophysics and The Howard Hughes Medical Institute, University of California San Francisco, San

Francisco, California 94143, USA2Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel.

3 Planck Institut fiir Entwicklungsbiologie, Spemannstrasse 35, 7400 Tubingen, FRG

'Present address: Department of Biological Sciences, Carnegie Mellon University, Box 194, Pittsburgh, PA 15213, USA

t Author for correspondence

Summary

The first event of Drosophila gastrulation is theformation of the ventral furrow. This process, whichleads to the invagination of the mesoderm, is a classicalexample of epithelial folding. To understand better thecellular changes and dynamics of furrow formation, weexamined living Drosophila embryos using three-dimen-sional time-lapse microscopy. By injecting fluorescentmarkers that visualize cell outlines and nuclei, wemonitored changes in cell shapes and nuclear positions.We find that the ventral furrow invaginates in twophases. During the first 'preparatory' phase, manyprospective furrow cells in apparently random positionsgradually begin to change shape, but the curvature ofthe epithelium hardly changes. In the second phase,

when a critical number of cells have begun to changeshape, the furrow suddenly invaginates. Our resultssuggest that furrow formation does not result from anordered wave of cell shape changes, contrary to a modelfor epithelial invagination in which a wave of apicalcontractions causes invagination. Instead, it appearsthat cells change their shape independently, in astochastic manner, and the sum of these individualchanges alters the curvature of the whole epithelium.

Key words: Drosophila, gastrulation, epithelial folding,furrow formation, time-lapse microscopy, fluorescentmarkers.

Introduction

During the development of an organism, many organsand other complex structures are created by the foldingof epithelial sheets. The formation of the germ layers ingastrulation is the earliest example during embryogen-esis. The first morphogenetic process during Drosophilagastrulation is the formation of the ventral furrow,which leads to the invagination of the mesoderm(Poulson, 1950; Sonnenblick, 1950). Drosophila ventralfurrow formation is an example of epithelial foldingthat is especially well suited for histological, cellbiological and genetical analysis. Compared with otherepithelial foldings, it is simple and quick. The process,which takes less than 20min, turns an epithelium ofmorphologically identical cells into an invaginated tubeof cells. No cell division occurs during this period. Likeother epithelial invaginations (reviewed in Ettensohn,1985; Schoenwolf and Smith, 1990), ventral furrowformation involves cell shape changes (Turner andMahowald, 1977, Leptin and Grunewald, 1990). Studies

on fixed embryos show that a population of prospectivemesoderm cells constrict at their apical ends and theirnuclei migrate basally (Leptin and Grunewald, 1990).Apical constriction of cells has been proposed to be adriving force for epithelial invaginations (Burnside,1973), and computer models suggest that a wave ofconstrictions transmitted from cell to cell might besufficient to produce a furrow (Odell et al. 1981).However, it is not possible to determine the temporaland possible causal relationships of the events duringepithelial invaginations from studies on fixed embryos.We therefore examined living embryos using three-dimensional time-lapse fluorescence microscopy(Hiraoka et al. 1989; Minden et al. 1989).

At the cellular blastoderm stage, the Drosophilaembryo consists of an epithelial layer of ~6000columnar cells (and ~40 spherical pole cells. Zalokarand Erk, 1976; Foe and Alberts, 1983). Whencellularization is complete, a band of cells (~20 cellswide and ~60 cells long) invaginates to form the ventralfurrow. This band is subdivided into two populations: a

366 Z. Kam and others

central population (8-10 cells wide), whose nucleimove basally and whose apical sides constrict, and aperipheral population (4-5 cells wide on either side ofthe central population) which follows the centralpopulation into the furrow without these shape changes(Leptin and Grunewald, 1990). We have concentratedour analysis here mainly on the behaviour of the centralcells, whose shape changes we believe to have an activerole in ventral furrow invagination.

Materials and methods

Preparation of embryosDrosophila melanogaster (strain Oregon R) embryos weredechorionated and mounted ventral side up with double-sidedtape (Scotch no. 665) on oxygen permeable teflon membrane(see below). The embryos were dehydrated slightly, coveredwith 700 weight halocarbon oil (HC Co. Hackensack NJ) andinjected as described (Minden et al. 1989). Calf thymushistones H2A and H2B were prepared as previously described(Minden et al. 1989) and injected at a concentration ofO^mgml"1 into the embryo at nuclear cycle 9 to 10.Fl-dextran (70000Mr, Molecular Probes, Eugene OR) atlmgml"1 was injected into the perivitelline space shortlyafter the Rh-histone injection. Recordings were done atapproximately 22 °C.

Microscopy and image and data processingLow light level images were recorded with a cooled, scientificgTade charge coupled device (CCD) camera operated by aself-contained computer workstation. Electronic shutters,focus position and fluorescence filter selection were alsocontrolled by the computer workstation. In order to focusmore than 30 /xm into the embryo at high magnification, it wasnecessary to image directly through the halocarbon oil inwhich the embryo was immersed rather than through acoverslip. The embryos could not be mounted on a glasscoverslip because they became anoxic when placed under themicroscope objective. Instead, they were mounted ventralside up on a thin oxygen permeable teflon membrane (YSIhigh sensitivity film, Yellow Springs Instruments, YellowSprings, OH) stretched over a specially designed platform.Under these conditions, the embryos developed normally atthe same rate as control embryos. A Zeiss PlanNeofluarx 63/1.2 water coverslip correction objective (Zeiss 461832)was used. The correction ring was turned to 0.22 mm to matchthe index of refraction of 1.414 for the halocarbon oil, asdescribed by Hiraoka et al. 1990. Ten optical sections, 3/flnapart, each taken in both fluorescein (0.2 s exposure) andrhodamine (0.1s exposure) channels (20 images altogether),were recorded for each time point. Because of the largedistances between focal planes, fluorescent background wassubtracted by image enhancement filters rather then bydeconvolution. The large dynamic range of the data (12 bits)allows such enhancement even for the high backgroundcaused by the large amount of out-of-focus fluorophores.

The nuclei in Figs 2 and 4 are modelled as ellipsoids withtheir centres, axial ratios and tilt angles calculated from theraw data as first and second moments of the correspondingvolume defined by histone fluorescence.The nuclear volumeparameters were automatically computed by an adaptation ofthe 2D ridge algorithm of Soferman and Shamir (Soferman,1989). The cell outlines were drawn using the PRISM 3Dmodelling software (Chen et al. 1990) by connecting theFl-dextran marked vertices between cells.

Results and discussion

To observe nuclear movement and cell shape changessimultaneously, two fluorescent probes were injectedinto the same embryo. Nuclear behaviour was observedby injecting rhodamine-labelled histones (Rh-histones)into embryos during the nuclear division cycles 9 and10. The Rh-histones are incorporated into chromatinduring the subsequent cycles of DNA replication(Minden et al. 1989). Cell outlines were visualized byinjecting fluorescein-dextran (Fl-dextran) into thespace between the plasma membrane and the vitellinemembrane. The Fl-dextran diffuses throughout theperivitelline space and reveals a bas-relief of the surfaceof the embryo (Warn and Magrath, 1982). Depressionsand spaces between cells appear brightly fluorescent,while the areas where the dextran is excluded from thecells appear as dark regions. The Fl-dextran is carriedinto intercellular spaces as the plasma membranesextend down between nuclei during ceUularization.

Two colour, three-dimensional data sets were ob-tained by alternately recording fluorescein and rhoda-mine images over 10 optical sections at 3 fjm incrementsonce every minute until the ventral furrow had formed.Fig. 1 shows a sample of these data with alternatingrows of Fl-dextran and Rh-histone images, each pairof images taken at the same focal plane. The columnsrepresent four time points at 5, 20, 24 and 28min afterthe beginning of the recording. Furrow formationbegan at approximately lOmin in this experiment. TheFl-dextran is found above the apical surfaces (Fig. 1,1A) and at the interstices of the polygonal cells (Fig. 1,3A and 5A). The leading edge of the ceUularizationfurrow appears as a crown-like ring of fluorescencearound the base of each cell (Fig. 1, 7A). Theinterstitial Fl-dextran signal persists throughout gastru-lation, while the crown-like rings disappear at thecompletion of ceUularization (Fig. 1, compare 7A and7B). All analysis shown in this paper was derived from asingle recording, but other experiments gave the sameresults.

The first visible cell shape change is the flattening ofthe hemispherical apical cell surfaces, seen as theaccumulation of bright patches of Fl-dextran aboveindividual cells (Fig. 2A; see arrows). The correspond-ing Rh-histone image 6/mi below the surface showsthat these patches lie above cells whose nuclei havebegun to migrate basally. The brightly labelled chromo-centres at the top of the nuclei are visible in this plane ofsection, while the neighbouring nuclei are seen at mid-section (Fig. 2B, see arrows). Computer models ofthree cells tracing cell outlines and nuclear positions areshown in Fig. 2C-E (these three cells were among thefirst to flatten their apical surfaces). For clarity, thenuclei are shown as ellipsoids, whose centres, dimen-sions and tilt angles are calculated from the histoneimages (see figure legend). Initially, the nucleus is at theapical side of the cell, and the cell is narrower at itsbasal end. The average ratio of apical to basal cross-sectional area is 1.6±0.2 (n=20 cells). The nucleidescend at an average rate of approximately

Cell shape changes in Drosophila gastrulation 367

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Fig. 1. A montage of optical sections ofa developing ventral furrow. Column Ashows a time point late incellularization, 5min after the beginningof the recording. The first signs ofventral furrow formation were visiblebetween 8 and lOmin. Columns B-Drepresent the 20, 24 and 28min timepoints, ending at the closure of thefurrow. The diagram at the top of eachcolumn (based on sections of fixedembryos; Leptin and Grunewald, 1990)shows the shape of the ventral furrowin the developing embryo (ventral sideup), at similar stages as the fluorescentimages below. Four focal planesseparated by 6,um are shown, each pairof rows representing Fl-dextran(labelled D at the right margin; rows1,3,5,7, ) and Rh-histone (labelled H;rows 2,4,6,8) images at the same focalplane. The Rh-histones label nuclei.The chromocentre appears as aparticularly bright region at the apicaltip of each nucleus. Dextran is confinedto the extracellular space. On thesurface of the embryo it marks celloutlines, until the apical surfaces beginto flatten (row 1). It accumulates overareas where the cell surfaces flatten andover the invaginating ventral furrow(columns B and D). Deeper in theepithelium, the dextran is found mainlyat the vertices of the hexagonal cells.No nuclei are initially visible at thelowest level (row 8), but as theydisappear from the top level (row 2),they begin to appear at the lowestlevel.

368 Z. Kam and others

Fig. 2. (A) The Fl-dextran distribution at the surface of theembryo at the first sign of cell shape changes. Fl-dextranaccumulates in the space created by the flattening of theapical surfaces of individual cells (arrows). (B) Thecorresponding Rh-histone image at 6,um below the cellsurface shows that the nuclei have begun to move basally(arrows) in those cells whose surfaces have flattened, andtheir chromocentres are visible in the focal plane shown.Although the more lateral cells have not flattened, theirnuclei are also sectioned at the level of the chromocentresbecause of the curvature of the surface of the embryo.(C-E) Position of the nucleus and contours of threeindividual cells from the centre of the ventral furrow at 2 minintervals. The nuclei are represented as ellipsoids, with theircentres and tilt angles calculated from the histone images asdescribed in Materials and methods. The separation betweenfocal levels of the outlined polygons is 3/jm. In order to givea clearer image, the scale on the y axis was halved. Timepoints: A and B: 12 min. C: 13, 15, 17 and 19min. D: 15, 17,19 and 21 min. E: 11, 13, 15, and 19 min.

2.5/anmin * (but not always continuously). As thenucleus moves away from the top of the cell, the apicalend contracts, and when the nucleus reaches the basalend of the cell, the cell is wider at its base than at theapex (average ratio of apical to basal cross-sectionalarea is 0.7±0.2; n=10 cells). The nuclei do not changetheir orientation within the cell during basal movement(as illustrated by the position of the chromocentres).Occasionally, the apical surface expands in cells that areadjacent to those whose apices have contracted. It is notpossible to discern whether nuclear movement and cellshape changes are caused by active contraction of theapical surface, or by a release of nuclei from the apicalsurface and passive compression of the cell. In somecases (Fig. 2C is an example), we see the nucleusbeginning to move basally in the absence of visibleapical constriction, suggesting that nuclear migrationmight not depend entirely on apical constriction.

The above cellular changes do not happen simul-taneously in all cells of the central population. Agraphical representation of the behaviour of 40 nuclei isshown in Fig. 3. The position of each nucleus along theleft-right axis of the embryo is projected onto thehorizontal axis for each time frame recorded, and thevertical axis represents time. Images of five time pointsare shown alongside the graph, with four representativetraces to demonstrate how the traces were constructed.Each trace terminates with a circle at the time that thenucleus moves below the plane of focus. For the cells ofthe central population, this represents the basalmovement of the nucleus within the cell (the approxi-mate edges of the central region are marked by trianglesin Fig. 3). For the peripheral cells, the disappearance ofnuclei corresponds to the movement of the whole cellinto the furrow. Two stages of ventral furrow formationcan be distinguished. During the first stage (min 10 tomin 22 in Fig. 3), more than half of the nuclei in thecentral population gradually move basally. There islittle movement of the lateral nuclei towards the centreof the furrow, and only a moderate change in thecurvature of the cell sheet. During the second stage, theremaining nuclei of the central population move basallyalmost simultaneously and the furrow deepens quickly(the Fl-dextran can be seen at a depth of 18 ̂ m; Fig. 17D). The peripheral cells now move laterally towardsthe furrow at a rate of about 10 fim min"

1 and fold overthe invaginated central population. The transition fromthe first to the second stage could either be due to theactivation of new cellular processes, or it could simplybe a mechanical collapse of the cell sheet as aconsequence of the continuing cellular processes of thefirst stage.

To give an integrated view of the data, Fig. 4 shows acomputer-generated three-dimensional reconstructionof the position and orientation of nuclei. Before thebeginning of ventral furrow formation, the nuclei areregularly aligned at the surface of the embryo(Fig. 4A). In Fig. 4B, many nuclei of the centralpopulation in apparently random positions have movedaway from the apical surface (highlighted nuclei havemigrated more than 6/im from the surface). Four

Fig. 4. A model of the positions of nuclei at four points of ventral furrow formation, derived from the same set of data asthose shown in Fig. 1. The nuclei are presented as ellipsoids with their centres and tilts computed from the histone imagesas in Fig. 2. Nuclei that have migrated away from the apical surface are shown in red. The time points are 1, 19, 23 and26min.

Cell shape changes in Drosophila gastrulation 369

15 30 45 60

Left-Right Axis (microns)

Fig. 3. Movement of nuclei during ventral furrow invagination. The positions of 40 nuclei in the region of the ventralfurrow are projected onto the x-axis and their movement traced over time. The y-axis indicates time, where t=0 is thebeginning of the recording. Since a strip of cells of approximately 12 cell diameters along the anterior-posterior axis of theembryo was evaluated, several cells were projected onto the same starting point. Centres of the nuclei are traced over timeat one focal plane 6jum below the surface. The trace is terminated by a circle at the point at which the nucleus disappearsfrom the focal plane. The approximate borders of the central population are marked by triangles on the x-axis. The ventralmidline is indicated by the open arrow. The dashed line indicates the transition between the first and second stages. Imagesof five time points (min 10, 14, 18, 22 and 26) are shown alongside the graph. Four representative traces demonstrate howthe traces were constructed.

minutes later, the majority of the nuclei in the centralpopulation have moved basally (Fig. 4C), while at thesame time the epithelium begins to invaginate, as seenby the accumulation of Fl-dextran in an intense bandover the furrow (Fig. 1, 1C). Subsequently, theperipheral population of cells folds over the invaginatedcentral population. The chromocentres of the periph-eral population remain at the apical surface and pointtowards the furrow (Fig. 1, panels 2D, 4D, 6D, and8D).

The order in which cells begin to change their shapeduring the first phase of ventral furrow formation isinconsistent with models that postulate a wave ofcontractions propagating from cell to cell as thetriggering event for invagination (Odell et al. 1981).Others have also noted an irregular appearance of cellsin invaginating epithelia, consistent with a stochasticinitiation of shape changes (Hardin and Keller, 1988;Schoenwolf and Franks, 1984). Since, in the ventralfurrow, neighbours of early constricting cells do notnecessarily constrict sooner than other cells in theepithelium, it appears that there are also no small localwaves of apical constrictions (however, an extensivestatistical analysis would be required to exclude themcompletely). Instead, our results suggest that the initialcellular events in furrow formation are part of anindependent program of activity of each cell in thecentral population. Thus, the question of how the

ventral epithelium begins to invaginate is reduced to theanalysis of how individual cells change their shape. Thetechniques described here will be useful in complement-ing genetic, and biochemical approaches to answeringthis question.

We thank Bruce Alberts, Daniel St Johnston and EricWieschaus for critical reading of the manuscript. J.S.M. is aLucille P. Markey scholar and his work was supported by agrant from the Lucille P. Markey Charitable Trust.

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(Accepted 21 March 1991)

Note added in proof

Observations on fixed embryos reported by Sweeton etal. {Development 112, 1991 in press) also suggest aninitial slow stochastic and a later fast phase of ventralfurrow formation.