Biol Cell (1991) 72, 15-23
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© Elsevier,Paris
Review
Genetic analysis of the cellularization of the Drosophila embryo F r a n f o i s S c h w e i s g u t h ! . , A l a i n V i n c e n t 2, J e a n - A n t o i n e L e p e s a n t a** zInstitut Jacques-Monod, CNRS et Um'versitd Paris 7, 2, place Jussieu, 75251 Paris Cedex 05; 2 Centre de Recherche de Biochimie et de Gdndtique Cellulaires. CNRS. 118, route de Narbonne, 31062 Toulouse Cedex, France (Received3 January 1991; accepted 7 May 1991)
Summary - The synchronous ceHularizationof the Drosophila embryo at the blastoderm stage provides a unique system for studying the molecular mechanisms involved in cytokinesis, using genetical and biochemical approaches. The cellularization process requires the major components of the embryonic cytoskeleton that are deposited into the egg during ongenesis. Genetical analysis indicates that it requires also the products of additional maternally-acting genes, as well as that of a limited set of zygotically-actinggenes. The cellularization defective phenotypes associated with small deficiencies uncovering these latter loci reveal specific steps within this complex process. The molecular analysis of these genes will ultimately provide meaningul insights into the normal process of cellularization. Among them, the serendipity a gene encodes a membrane-associated protein, which is exclusivelyacculumated during cellularization, and is required for the reorganization of the microfilaments as the onset of cellularization. Drosophila I cdlulm'inflon I cyllokJnesisI i¢tin I cytoskdetonI bl~oderm I s~rend/p/O,a
Introduction In animal cells, the cleavage of one cell into two cells at the end of mitosis, or cytokinesis, proceeds by constriction of the mother cell. Many decades of experimental research have provided different conceptual frameworks to account for the localized formation during telophase of the cleavage furrow and for its subsequent inward progression [34, $6]. It is now widely accepted that the mitotic apparatus somehow determines the furrow's position by inducing regional alteration of surface properties [34]. Thereafter cleavage activity itself relies upon a transient dynamic structure, the contractile ring, first described by Schroeder [39] as a belt of aligned microfllaments. Although the ultrastructural elements essential for cytokinesis have been identified long ago, the molecular mechanisms that regulate cytokinesis are still largely unknown (reviewed in [40]). In spite of great progress in the biochemical identification of the factors that modulate contractile proteins activity [26, 32], the mechanisms that control formarion, localization, maintenance and activity of the contractile ring during furrowing have not yet been determined. A molecular genetics approach might prove useful to evaluate the roles of the major cytoskeleton components, as well as to identify new molecules important for its regulation. Among the organisms with elaborate genetics, the fruit fly shows a striking form of cytokinesis early in development that consists of the cellularization of the syncytial embryo. A network of multiple cleavage furrows concertedly pushes into the syncytial Drosophila embryo to form simultaneously a monolayer of about 6 000 epithelial-like cells. The aim of this review is to summarize our current knowledge of the mechanisms involved in the cellulariza-
* Presentaddress:Departmentof Biology,B-022, Universityof California, San Diego, CA 92093 La JoHa, USA ** Correspondenceand reprints
tion process, and to emphasize more particularly the importance of molecular genetics in the analysis of this process. Cdlulsflzsflon of the wild.type Drosophila embryo The early Drosophila embryo initially develops as a syncytium. The first 13 mitotic divisions occur synchronously in the egg cytoplasm without cytokine#is [10]. The nuclei are initially located in the center of the egg. The majority of the nuclei migrate stepwiseto the cortical cytoplasm during cycles 7-10. Only 26 nuclei out of a total of 128 remain located in the center of the egg. These "viteilophag.e" nuclei stop dividing, become polypoid and never give me to cells. The 10-12 nuclei that reach the posterior pole of the embryo are incorporated into polar buds at cycle 9. These polar buds pinch off at the end of mitotic cycle 10 (80 man after fertilization). This process leads to the formation of the first cells, the pole cells which are precursors of the germ Hne cells. The rest of the nuclei are the somatic nuclei. They distribute as a uniform monolayer at the cortex of the egg and undergo three more nuclear divisions (cycles 11 to 13) to form eventually a cortical layer of about 6000 somatic nuclei. Each nucleus is surrounded by its own individualized cytoplasmic domain despite the fact that it is localized in a syncytium [20]. During each interphase of the mitotic cycles 11-13, this cytoplasmic domain forms a cap over the nucleus, which appears as a bulge at the surface of the embryo [58]. Early in metaphase, each cap extends and spfits into two daughter caps prior to nuclear division |53]. A membrane invagination appears around each chvlding nucleus at metaphase. It regresses during telophase while the two caps associated with the daughter nuclei reft',Tm, so that the cleavage furrow formed during nuclear ~:y:~;!e13 regresses prior to cellularization. These membrarie iiwaginations are referred to as transient cleavage furrows []3]. Cellularization occurs simultanz~,~u;ly across the whole
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F Schweisguth et at
surface of the egg at the end of mitosis 13 and proceeds during the interphase of cycle 14 Coetwcvn 120 to 170 rain after fertilization) [10]. The plasma membrane invaginates down from the overlying surface between adjacent nuclei (fig 1). The invaginating membranes form an hexagonal array of cleavage furrows as viewed from above. All the cleavage furrows advance synchronously into the cortical cytoplasm, first at a slow rate during the so-called "slow phase" (between 120 and 155 min), and then more rapidly during the next 15 min of the "fast phase" of cellularization [58]. At the base of the cleavage furrow, the apposed membranes separate to form the furrow canal [12]. This structure appears as a triangular pocket on transverse sections, and as a circle on tangential view. The presence of the furrow canal distinguishes the cellularization furrow from the transient cleavage furrow that forms during each interphase of the nuclear cycles 11-13. The origin of the membranes that contribute to the increase in the plasma membrane surface remains unclear. Formation of multiple villous projections is observed prior to cellularization [12, 50]. These plasma membrane folds persist during the "slow phase" of cellularization. Fusion of newly synthesized endomembranes may contribute rather to the formation of the lateral membranes during this first phase [38]. The apical surface only flattens at the onset of the "fast phase", as if the plasma membrane accumulated in the villous projections had been pulled down in the cleavage furrow. It is supposed that this flattening process provides about half of the membrane surface required to enclose the blastoderm somatic cells [12]. Therefore, the difference in the rate of membrane inward progression between the slow and fast phases of cellularization may in part reflect the different mechanisms of membrane formation [12].
.. .
:
..
.
Cycle 13, telophase
Cycle 14, early phase I
Cycle 14, mid-cellulartzatlon
Cycle 14o late phase II
Fig 1. Schematic representation of the cellularization of the
D melanosasterembryo. Transverse views of an embryo from
telophase of mitotic cycle 13 until the end of cellularization. Nuclei are darkened. Stipplingindicates Foactindistribution. Centrosomes and microtubules are line.drawn. Stages are indicated below each view.
The Drosophila contractile ring network Accumulation of electron-dense filaments has been observed beneath the furrow canal during cellularization, forming a ring-like structure [12, 21]. This material progressively accumulates in a depressed zone of the surface plasma membrane [12, 21]. This circular depressed zone appears at the end of the telophase of the 13th syncytial division. Its diameter is defined by the midline of the underlying mitotic apparatus and the position where the cycle 13 transient cleavage furrow was previously located [12, 21]. This fibriUar material is composed of abundant filaments of actin [54], which are bidirectionally arranged [21], and of myosin II [59]. F-actin is found associated both with the lateral membranes, where it forms a roughly hexagonal network, and with the apical membrane folds during the first phase of cellularization. Small F-actin aggregates are also present within the cell below the cellularization front [54]. F-actin is no longer detectable in the cap region after the apical plasma membrane has flattened during the fast phase [54]. A number of actinbinding proteins are also specifically associated with the filamentous ring-like structure described above, or with other distinct subsets of actin filaments [29]. During membrane invagination, this ring-like structure remains associated with the furrow canal. Direct contacts between adjacent rings are commonly observed [54]. These interconnections hold all the rings together in a continuous network. The ring's diameter remains roughly constant during the slow phase of cellularization. It reduces in size during the fast phase of cellularization, so that each ring appears to be separate [54, 59]. Contraction of the ring pulls the membrane of the canal, which considerably enlarges, and results in the formation of the basal membrane of the epithelial.like blastoderm cells. After cellularization, a small bridge, which connects the cytoplasm of each cell with the yolk, persists until germ-band elonga. tion. The ring-like structure completely disappears at the end of cellularization. Local injections of cytochalasin [8, 10, 61], which depolymerizes F.actin, or of anti-myosin antibodies [24] block both the formation of this ring-like structure and the cellularization near the point of injection. These results indicate that the intactness of the ring-like structure described above is required for cellularization. The term "contractile ring network" has been proposed to describe this integrated array of ring-like structures in the Drosophila embryo [55], because they appear similar in composition, structure and function to the contractile rings of dividing cells as discussed below. The cellularization process is common to all insect species. It generally follows the description given above for D melanogaster. However many apterygote eggs, such as the Collembola eggs, do not develop as a syncitium, since early nuclear divisions are followed by complete cytokinesis [17]. Variations have also been reported in certain hemimetabolous insects. In lsoptera and Cheleutoptera, cortical nuclei distribution is highly polarized in the differentiating syncytial embryo. The nuclei are closely packed near the posterior pole, while being widespread elsewhere. By contrast to the Drosophila uniform cellular blastoderm, which is formed by the synchronous cellularization of a monolayer of evenly distributed nuclei, the differentiated cellular blastoderm is formed by the progressive aggregation of cells from the anterioly located extraembryonic region to the embryonic primordium, the latter resulting from the localized cellularization of the nuclei
Cellularizationof the Drosophila embryo clustered near the posterior pole [3]. The presumed differences between the invagination of the plasma membrane in the extra-embryonic versus the embryonic primordium has not been analyzed in these species. It would be of interest to analyze the formation of the contractile network at the borders of the embryonic primordium in these species. Functional similarities between the contractile ring network of the Drosophila embryo and the contractile ring of dividing cells The contractile ring in dividing animal cells is mainly composed of actin and myosin filaments, arranged bidirectionally in a transient ring structure. It apparently forms primarily through the reorganization of pre-existing cortical actin filaments that directionally move towards the equator region of the dividing cell [5, 6]. Sliding interactions between actin and myosin f'daments are generally thought to provide the furrowing force [40]. Indeed, injection of cytochalasin, or of anti-myosin II antibodies, in dividing cells results in the disruption of the contractile ring and in the blockage of the inward progression of the cleavage furrow (see [40] and references therein). Likewise, deletion of the single myosin II gene in Dictyostelium has provided clear evidence that myosin II is an indispensable motor for cytokinesis [11]. The Drosophila contractile ring network therefore appears highly similar in composition and structure. However, there are no clear data relevant to the mechanism of assembly of actin and myosin filaments into a contractile ring network. It is not known whether de novo assembly of unpolymerized subunits onto nucleation sites accounts for the contractile ring network formation, or whether conical pre-existing fdaments migrate from the cap towards the depressed zone where the contractile rings appear [21], as described for cytokinesis [$, 6, 21]. It has been suggested from the description of the F-actin organization by fluorescence staining that the actin filaments stocked in cytoplasmic aggregates become progessively part of the contractile rings [54]. This hypothesis is consistent with the mechanisms proposed for contractile ring formation in dividing cells by Cao and Wang [5, 6]. Likewise it is not yet clear whether actin and myosin filaments provide the furrowing force during cellularization in a manner similar to cytokinesis, or whether they are required to stabilize the network across the whole surface of the egg during cellularization. The results obtained by injections of cytochalasin and of anti-myosin II antibodies, ie local inhibition of cellularization, are consistent with the hypotheses that the contracting ring network acts as a motor for cellularization or as a structural passive element. The structural similarities presented above between contractile rings involved in cellularization and cytokinesis argue in favor of the assumption that the contractile ring network plays a motor role. These similarities appear even more striking if one considers the unilateral, or heartshaped, cleavage described in coelenterate eggs [35]. In this variant of cytokinesis, the mitotic apparatus is excentrically located at the animal pole. The furrow only appears in the animal pole surface, and proceeds unilaterally towards the vegetal pole. In the Drosophila embryo also, the mitotic apparatus is localized in the cortic',d cap, close to the apical surface, which by analogy corresponds to the animal pole surface. The yolk-rich center of the egg would be then considered analogous to the vegetal pole of the
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coelenterate egg. However, these two cleavage figures still differ by the relative orientation between the cleavage furrows and the contractile rings. The contractile ring is located in the same plane as the advancing cleavage furrows during cytokinesis, even in the case of the unilateral cleavage of coelenterate egg [40], while the Drosophila contractile ring network is oriented perpendicularly to the invaginating furrow membranes. This difference in geometrical arrangement indicates that the role of the contractile ring network during cellularization cannot be directly inferred from the one proposed for the contractile ring in dividing animal cells. The contractile activity of the F-actin meshwork might be required only at the end of cellularization to pinch off cells. During the fast phase of cellularization, each contractile ring becomes progressively reduced in size as the cleavage furrows approach the inner yolk mass [54]. Sliding interactions between actin and myosin filaments are thought to be responsible for this reduction in size, which results in furrow canal enlargement and basal membrane formation [54]. Because contraction of each ring correlates with the formation of the basal membranes proceeding in the same plane as the one defined by the contractile ring network, it has been suggested that additional forces may be required to either "pull" or "push" down the cleavage furrow in a direction orthogonal to the one defined by the contractile ring [55]. The contractile ring network might then be essentially required during the first slow phase of cellularization to ensure the cohesiveness of the entire matrix as a structural or tension bearing element. These speculative considerations should make it clear that the mechanism of cellularization is far from being understood. The phenotypical analysis of cellularization defective mutations may help us to dissect the discrete mechanisms of this process. Phenotypes of cellulaflzatlon mutant embryos Mutations that affect cellularization have been recovered in genetic screens for female-sterile loci [36, 42, 60], and for zygotically required loci [28, 58]. Cellularization of the syncitial blastoderm embryo thus depends upon both maternal and zygotic factors. Indeed, most gene products required for early embryogenesis are synthesized during oogenesis and deposited into the egg [27]. In addition, specific genes are zygotically transcribed prior to cellularization, as early as nuclear cycle 11 [9, 44], thereby accounting for the zygotic contribution. T:le phenotypes associated with these two classes of mutations are detailed in the following paragraphs. Maternal-effect mutations
About 25 maternal-effect nmtations have been isolated that affect the cellularization process [36, 41, 42, 60]. In most cases, however, their cellularization phenotype has not been yet analysed in great detail. Thefs(l)1459 ~ mutation is among the best described [60]. At high temperature, the development of embryos laid by females homozygous for thefs(l)1459 ts mutation is specifically arrested at the cellular blastoderm stage. The thermosensitive period of this mutation is restricted to this developmental stage. Electron micrographs show that cell membranes do not invaginate in fs(1)1459 ts embryos at the time when the nuclei start to elongate. The furrows remain short and irregular. No furrow canals can be detected at the base of
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the membrane furrows. The nuclei eventually migrate deep into the periplasm. Interestingly, the fs(1)1459 ts embryos are in many aspects similar to cytochalasin injected embryce, suggesting a defect in microfdament-based activity. As a general rule, the cellularization defects associated with zygotic or maternal-effect mutations are distributed uniformly across the whole surface of the embryo. A few maternal-effect mutations (mat(3)3 and mat(3)6 [36]; torso and trunk [41]; maternal effect of I(l)pole hole [11]) exhibit |ocaliT~i defects and constitute notable exceptions. The mat(S)3 mutation results in a failure of cell formation on the dorsal-posterior region. Conversely, in matO)6 mutant embryos, cellularization is detectable only at poles where complete cells form [36]. In both mutants the noncellularized region presents extensive defects: the nuclei move away from their apical position in the cortical cytoplasm, which is itself invaded by yolk. The initial defects detectable in these mutant embryos have not yet been analysed in detail. Mutations in the torso, trunk and I(l)pole hole genes, which primarily affect abdominal pattern formation, are three other interesting exceptions. Loss of function alleles of these genes shows localized cellularizatinn defects at the posterior pole [1, 41]. The torso gene activity is specifically restricted to the posterior pole [7]. The torso gene encodes a membrane receptor with a tyrosine kinase cytoplasmic domain [47]. The torso protein is thought to regulate the activity of ser/thr kinases involved in the induction of posterior pole development [2, 31]. One of these kinases is encoded by the l(l)pole hole gene [1, 45]. The observation of localized defects, and the fact that cellularization appears to be a uniform process across the whole surface of the embryo, raise an apparent contradiction. In order to reconcile these contradictory terms, we propose that protein phosphorylation is a key control mechanism of cellularization across the whole surface of the embryo, and that certain mutations affecting a phos. phorylation cascade in a specific region of the embryo are able to interfere with the ceilularization process in that same area. Then, the fact, that cellulariLation defects are detectable in a specific region of the embryo surface does not necessarily imply that the mechanism of cellularization is not identical in all somatic cells. It remains to be determined whether the localized defects observed in mat(S)3, mat(S)6 and trunk embryos are also secondary consequences of an altered process of pattern formation. Zygotically required loci
The Drosophila genome has been essentially saturated with zygotic lethal mutations, and those resulting in specific cuticular phenotypes have been genetically analysed [18, 30, 57]. None of the zygotic lethal mutations isolated in the course of this extensive screen resulted in a cellularization phenotype. It was however already known that synthesis of zygotic factors is required for cellularization, as shown by inhibiting de novo transcription through injection of ~-amanitin in cleave~e-stageembryos, which results in abnormal cellularization [4]. One possible explanation for the non-isolation of zygotic lethal mutations affect. ing cellularization is that no cellularization defective mutants exhibited a reproducible cuticular phenotype, and the putative cellularization mutations were therefore discarded by these authors [18, 30, 57]. Another possibility is that, among the putative zygotic cellularization mutations, those which result in the formation of an abnormal number of cells do not result in embryonic lethality. Indeed, embryos
laid by females homozygous for the daughterless-abo-like (dal) maternal-effect semi-lethal mutation show an impressive ability to recover from a secondary cellularization defect [48]. These embryos cellularize with only about onehalf the normal number of cells, and 8~/e even develop to adulthood [48]. These remarks suggest that detection of cellularization phenotypes may require the direct observation of blastoderm-stage embryos. Therefore, the development of living embryos deficient for cytologically defined portions of the genome has been followed in order to identify genomic regions that are zygotically required for the cellularization of the embryo [28, 58]. Attached X-chromosomes, compound autosomes and Y translocations were used to generate these deficient embryos. After scanning the whole genome, only six chromosomal regions were identified that were associated with a unique cellularization phenotype when deleted. It can thus be predicted that each of these genomic regions contains at least one zygotic cellularization locus. In all cases, numerous other defects appear after cellul"anzation in these deficient embryos which finally die. For this reason, the phenotype associated with each deficiency, as detailed below and summarized in table I, corresponds only to the earliest detectable one. The establishment of the hexagonal F-actin network requires gene activity from three distinct genomic regions. Deficiencies encompassing these regions define the loci 6F-TA or hullo, 99D and 100 AC, so-named after their respective position on the cytological map of the polytene chromosomes. The phenotypes associated with the lack of hullo, 99D or 100AC gene activities are initially s!m_i!ar, with hullo defects being the most severe. The phenotype associated with 100AC deficiencies differs from the two others later in cellularization (see below). These pheno. types consist in erratic disruptions of the cytcekeleton early at mitotic cycle 14 (fig 2) leading to the formation of cells that contain multiple nuclei [28, 43, 46, $8]. These phenotypes suggest that the reorganization of the actin-myosin filaments into an hexagonal network prior to membrane invagination is regulated, but do not indicate whether this reorganization proceeds through directional migration of pre existing filaments or de novo filament polymerization at localized nucleation sites. It should also be noted that no specific abnormalities are detected at the syncytial blastoderm stage. Specifically, the transient cleavage furrows normally form at the interphases of mitotic cycles 11-13. This indicates that the formation of pseudocleavnge furrows during cycles 11-13 and of cleavage furrows during cellularization are partly under separate genetic control. The lack of 100AC p n e activity also results in deregulated contractile activity of the contractile ring network. Each contractile ring reduces in size before midcellularization, as the actin and myosin f'daments progressively contract, entrapping the nuclei at the cellularization furrow [28]. Although the cytoskeleton remains firmly anchored to the plasma membrane, the invagination of the latter is inhibited [28]. This phenotype suggests that the 100AC locus is required either to keep the contractile rings interconnected in an integrated network, or to regulate temporally the sliding interactions between actin and myosin filaments to prevent each ring from contracting before mid-cellularization. The plasma membrane invagination requires two other loci, named 26BF and 40AC. The analysis of their respective phenotype provides a functional distinction between the slow and fast phases of cellularization [28]. These two
Cellularizationof the Drosophila embryo
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Table I. Summary of the phenotypes associated with the zygotically-required loci. For each locus listed in the leftmost column, the corresponding ceHularization-defective phenotype is diagrammed. Plasma membrane is drawn as a line and nuclei are darkened. The arrow indicates the invaginafion rate of the plasma membrane, the thinner part corresponding to the initial slow rate, the thicker part corresponding to the following fast rate.
Locus
Chromosomal localization
References
nullo
6F-TA
[46, 58]
sly ~
99D4-8
[28, 43l
IOOAC
IOOAC
[28]
Formation of plurinucleate ceils and premature closure of contractile rings
26BF
26BF
[281
No membrane invagination during the slow phase
40AC
40AC
[281
No accelerated rate of membrane invagination during the fast phase
[28]
Disruptions in the conical localization of the somatic nuclei at an early stage of cellularization
71C-75C
71C-75C
Phenotype
Formation of plurinucleate cells
phases, initially defined by their different rates of membrane lnvngination [$8], can also be distinguished by their distinct genetic requirements. Deletion of the 26BF locus results in a lack of membrane invngination during a 40 rain period after completion of mitosis 13, a period during which the slow phase of cellularization normally takes place. Thereafter, the plasma membrane rapidly invaginates, at a rate similar to that of the fast phase of cellularization in wild-type embryo [28]. Cellularization is not uniformly completed in these embryos before gastndation. Conversely in the absence of 40AC gene activity, no fast phase is detectable. The plasma membrane continues to invnginate at a slow rate until gastrulation, which takes place at the correct time [28]. This clearly indicates that cellularization is a two-step process, and that the two phases are in part functionally independent. Finally, deletion of the locus 71-75C affects the apicalbasal distribution of the nuclei, as many nuclei sink into the interior of the embryo [28]. A s~dlar defect is observed following cytochalasin B injection [8] that results in the extensive reorsanization of both microfilaments and microtubules. In contrast to cytochalasin injected embryos, membrane invagination proceeds normally in embryos homozygous for a 71C-75C deficiency. This difference suggests that these two actin-based processes, ie the cortical localization of the nuclei and the invagination of the
plasma membrane, involve distinct specific factors, one of which is encoded by the locus 71C-75C. No specific mutation in any of these six loci has yet been Lqolated. Whether a mutation leading to the complete lack of a function in any one of these six zygotic loci is lethal to the embryo, or affects other morphogenetic processes is therefore not yet known. Related loci
There exist additional mutations that affect in a nonspecific manner the cellularization process. The shibire thermo-sensitive mutation (shits) is one well-documented example. In shP homozygous animals, endocytosis is inhibited at restrictive temperature. This inhibition in turn results in various developmental defects [33], including cellularization defects. When cellularizing blastoderm embryos are shifted at restrictive temperature, the furrow membranes are disrupted into numerous vesicles [49]. This suggests that shibire activity i~ required for a general function related to cycling of vesicles and/or to membrane stability. The last group of mutations, whose phenotypes are possibly related to cellularization, corresponds to cytokinesis defective mutations. Two such mutations, l(l)d degll and
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/ /
B
Fig 2. Cellularization defects associated with the 99D deficiency, Embryos homozygous for the deficiency 99D (Df(3R)X3F: [43]) (B, D and F) are compared to wild.type embryos (A, C and E). A and B. Sectionsthrough surface cortex reveal the irregular distribution of the nuclei in deficient embryos. C-F. Disruptions of the F-actin array, visualized on surface views (C and D) and side views (E and F) of whole-mount embryos using fiuorescently labelled phalloidin. Bars: 5 pro.
1(3) 7m.62, have been recognized among a collection of
larval and/or pupal lethal mutations as the ones producing highly polypoid cells in larval brain squashes [15]. The 1(33 7m-62 locus, or spaghetti-squash, has recently been shown to encode the myosin [I regulatory light chain [19]. It is not yet known whether these two loci are required for ceilularization.
The cellularization defective 99D locus is the blastodermspecific serendipity ~ gene The isolation of the maternal-effect mutations and the cytological mapping of the zygotically required loci provide the first steps for going beyond the description of the cytoskeletal components and moving on to the molecular identification of these factors.
The smallest deficiency that defines the 99D locus covers 9 cytological bands, and encompasses many transcription units [52], among which is the serendipity ~ (sty ~) gene [51], located at position 99D4-8. Transcription of the sty gene starts at nuclear cycle 1I, peaks at the onset of cellularization and rapidly decreases during the fast phase of cellularization ([44] and F Schweisguth, unpublished results). Sequence analysis of the encoded protein does not reveal any significant homology to known proteins [51]. Since the zygotic ceilularization phenotype associated with 99D deficiencies [28] appears at the onset of zygotic transcription, it probably results from the non-expression of a strictly zygotic and early expressed gene. The hypothesis that the cellularization phenotype associated with deficiencies encompassing the 99D region is due to the lack of sty ~ gene activity was tested by P element rescue experiments. Stable insertion of a functional sty ~ gene into
Cellularization of the Drosophila embryo the genome of a 99D deficient embryo is able to restore a normal cellularization process. The sty ~ gene activity alone is therefore sufficient to rescue the cellularization defects associated with the 99D deficiency, indicating that the sty • gene is required for cellularization [43]. The sty gene is the first gene to be identified that is specifically required for the cellularization of the Drosophila embryo. None of the other blastoderm-specific genes, isolated by differenti~ cDNA screening [25], is located on the polytene chromosomes map within intervals that define the cellularization defective zygotic loci. Using specific antibodies, the 58 kDa sry ~ protein has been detected in association with the folded apical membrane at the onset of cellularization, and with the furrow membranes during invagination. The sry ~ protein is specifically accumulated at the level of the furrow canal during cleavage formation [43], and appears to be part of the contractile ring (fig 3). This localization is similar to the ones given for the actin and myosin filaments [54, 59]. No sry ~ protein or transcripts are detectable after midgastrulation. During the whole fife-cycle of the fly, the sry protein accumulates for only one hour, indicating that normal cytokinesis does not require the presence of the sry ~ protein. The sry ot protein is also dispensable for pole cell formation at mitotic cycle 10. The phenotype of the 99D deficient embryos (fig 2) suggests that the sry a protein is required for the initial reorganization of the apical microfilaments into a regular hexagonal network. One simple hypothesis would then be that the sry ~ protein binds to F-actin. However, it is not among the 40 actin-binding proteins purified from early embryos by F-actin affinity chromatography ([29] and
21
C Field, personal communication), and no putative actinbinding motifs have been identified within the predicted sequence of the protein. Conclusions and prospects The genetic analysis of cellularization is still at an early stage. In most cases, the maternal-effect mutations still await an accurate phenotyr'." description. With regards to the zygotically-required loci, no specific mutations, as opposed to deficiencies, have yet been recovered. Nevertheless several conclusions may be drawn already. First, one of the most remarkable features of the phenotypes described above is their specificity for particular aspects of cellularization. Such specifities in phenotypes should make it possible to dissect the discrete mechanisms of this complex cellular process. This appears especially true for the zygotically-required loci. This could be simply due to a more detailed phenotypic ~atysis of the zygotic loci as compared to the maternal ones. Alternatively, one attractive possibility is that the zygotically-acting genes encode regulatory factors whose accumulation must be developmentally regulated, while maternally-acting genes mostly contribute to the accumulation of general factors, such as the major and ubiquitous components of the cytoskeleton. Also, none of these mutations specifically affect the timing of cellularization. Cellularization is never observed prior to cycle 14. The onset of cellnlarization does not depend upon nuclear density. As described above, ceilularization occurs normally at mitotic cycles 14 in dal- embryos,
Fig 3. Localization of the sty, protein in the contractile ring. Top view of the contractile ring network of a wild type mid-cellularizing embryo of D melanogasterrevealing the distribution of the sry • protein detected by indirect immunofluorescence using immunopurifled anti-sry • antibodies and rhodamine-conjugated secondary antibodies excited at 488 nm. This photograph was obtained using the × 40 objective on a Biorad MRC 100 confocal laser scanning microscope. Bar: 5 ttm.
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F Schweisguthet al
though there is only one-half the normal number of nuclei [48]. The timing of cellularization is also not tightly coupled to the transition between synchronous nuclear- cycles and spatio-temporally regulated mitosis. Indeed, cellularization be~i'ns at the proper developmental time even when an extra round of syncytial division is observed, as, for example, in maternal haploid embryos [9]. The maternal haploid mutation, originally named fs(1)572, was isolated as a maternal-effect mutation affecting cellularization by Zalokar et al [60]. In these embryos the 14th synchronous mitosis interrupts cellularization, causing developing cell membranes to partially regress. The mechanism that determines the onset of cellularization at the end of nuclear cycle 13, in a manner independent of the nuclear density and of the total number of synchronous syncytial cycles, remains unknown. Surprisingly, none of the ceilularization defective mutations isolated to date correspond to the single myosin II gene [23], or to any member of the cytoplasmic actin encoding mnltigene family [13, 14]. Two very different reasons may account for this observation. In the case of the myosin II gene, strong loss of function mutations would presumably results in cell autonomous lethality. This means that such mutations would surely affect cell vialibity during oogenesis, therebey preventing the production of fertilized eggs. This cell lethal effect might be expected to occur in germ line clones homozygous for such mutations, generated in heterozygous mothers. The problem differs in nature for the actin genes because the coding sequences of the two cytoplasmic actin genes are highly homologous [14]. Therefore a mutation in one of the two cytoplasmic actin genes might be compensated for by the other unmurated one, or by any one of the four other actin genes. This hypothesis assumes that none of the isoforms serves a highly specialized and non-compensable function during cellularization (see [37] for a discussion of the differential iso-actin utilization in nonmuscle cells). The phenotypical evaluation of the function of the actin and myosin filaments during cellularization and cytokinesis in Drosophila embryo awaits the isolation of conditional mutations in the myosin II and actin genes, such as thermosensitive mutations. In the case of the actin genes, the desired mutation has to be not only conditional but also "dominant negative", so that mutated actin gene products interfere with the activity of the wild-type products, through the incorporation of inactive subunits in multimeric filaments. These types of mutational events are expected to be rare, and have indeed not yet been observed. Nevertheless, the ability to manipulate in vitro the cloned genes and to reintroduce them into the Drosophila genome opens the way to the design of conditional dominant negative mutations, as discussed by Herskowitz [16]. Finally, in almost all of the zygotic and maternal-effect mutant embryos described above, pole cells form normally. Pole cell and blastoderm cells formation appear to be under separate genetic control. The mechanism of pole cell formation has not been dealt with in this review. The combination of classical genetics, molecular biology and biochemistry makes the cellularization of the Drosophila embryo an attractive model for the elucidation of fundamental questions on cytokinesis. The mechanism that determines the nuclear cycle at which cellularization occurs, the nature of the factors that determine the position where cleavage furrows will subsequently form, the way the contractile ring network is generated and maintained, remain largely unknown. Many cellularization defective loci, but certainly not all, have
been isolated. In an independent approach, a number of actin-binding and microtubule-associated proteins have been identified by affinity chromatography [22, 29]. These two approaches may ultimately converge to define a limited set of genes. Molecular and biochemical studies will then be required for a better understanding of the function of these genes. Until now these data remain preliminary, and any attempt to propose a model for the cellularization of the Drosophila embryo would still largely rely on studies of cytokinesis in cultured cells or in marine invertebrate eggs. Hopefully, the genes that regulate ceilnlarization will be cloned in the near future, the proteins they encode characterized and their role during cellularization and cytokinesis evaluated in vivo by genetical means. The molecular dissection of the cellularization process may then greatly assist analysis at the molecular level of the universal regulatory mechanisms involved in cytokinesis. Acknowledgments We thank E Wieschaus and DP Kiehart for preprints, C Field for communicating unpublished results, J Deutsch and H Brock for comments, G C~raud for help with confocal microscopy and A Kropfinger for revision of the manuscript. References
1 Ambrosio L, Mahowald AP, Perrimon N (1989)I(l)pole hole is required maternally for pattern formation in the terminal regions of the embryo. Development 106, 145-158 2 Ambrosio L, Mahowald AP, Perrimon N (1989) Requirement of the Drosophila raf homologue for torso function. Nature 342, 288-290 3 Anderson DT (1972) The development of hemimetabolous insects. In: Developmental systents: insects (Counce SJ, Waddington CH, eds) Academic Press, London, New.York, 9~- 163 4 Arking R, Parente A (1980) Effects of RNA inhibitors on the development of Drosophila embryos permeabilJzed by a new technique. J F.xp Zool 212, 183-194 5 Cap LG, Wang YL (1990) Mechanism of the formation of
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contractile ring in dividing cultured animal cells. I. Recruit. ment of pre-existing actin filaments into the cleaver fur. row. J Cell Biol 110, 1089-1095 Cap LG, Wang YL (1990) Mechanism of the formation of contractile ring in dividing cultured animal cells. !I. Cortical movement of microinjected actin filaments. J Cell Biol 111, 1905-1911 Casanova J, Struhl G (1989) Localized surface activity of torso, a receptor tyrosine kinase, specifies terminal body pattern in Drosophila. Genes Dew 3, 2025-2038 Edgar BA, Odell GM, Schubiger G (1987) Cytoarchitecture and the patterning of fushi tarazu expression in the Drosophila blastoderm. Genes Dew 1, 1226-1237 Edgar BA, Schubiger G (1986) Parameters controlling transcriptional activation during early Drosophila development. Cell 44, 871-877 Foe VE, Alberts BM (1983)Studiesof nuclear and cytoplas. mic events during the five mitotic cycles that precede gas. trulation in De~phila embryogenesis.J Cell Sci 61, 31-70 Fukui Y, De Lozanne A, Spudich JA (1990) Structure and function of the cytoskeleton of a Dictyostelium myosindefective mutant. J Cell Biol 110, 367-378 Fullilove SL, Jacobson AG (1971) Nuclear elongation and cytokinesis in Drosophila montana. Dew Bio126, $60-$77 FyrbergEA, CmldsteinLSB (1990)The Drosophila cytoskeleton. Annu Rev Cell Bioi 6, 559-596 Fyrberg EA, Mahaffey JW, Bond BJ, Davidson N (1983) Transcripts of the six Dwsophila actin genes accumulate in a stage- and tissue-specific manner. Cell 33, 115-123
Cellularization of the Drosophila embryo 15 Gatti M, Baker BS (1989) Genes controlling essential ceilcycle functions in Drosophila melanogoster. Genes Dev 3, 438-453 16 Herskowitz I (1987) Functional inactivation of genes by dominant negative mutations. Nature 329, 219-222 17 Jura C (1972) Development of apterygote insects. In: Developmental systems: insects (Counce SJ, Wadclington CH, eds) Academic Press, London, New-York, 49-94 18 Jfirgens G, Wieschaus E, NOsslein-Volhard C, Kluding H (1984) Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster III. Zygotic loci on the Xchromosome and fourth chromosome. Roux's Arch lX,v Biol 193, 296-307 19 Karess RE, Chang XJ, Edwards KA, Kulharni S, Aguilera I, Kiehart DP (1991) The regulatory light chain of nonmusde myosin is encoded by spaghetti-squash, a gene required for cytokinesis in Drosophila. Cell 65, 1177-1189 20 Karr TL, Alberts BM (1986) Organization of the cytoskeleton in early Drosophila embryo. J CellBio1102,1494-1509 21 Katoh K, Ishikawa H (1989) The cytoskeletal involvement in cellularizationof the Drosophila melanoguster embryo. Protoplosma 150, 83-95 22 KelloggDR, Field CM, Alberts BM (1989) Identification of microtubule-associated proteins in the centrosome, spindle, and kinetochore of the early Drosophila embryo. J Cell Biol 109, 2977-2991 23 Ketchum AS, Stewart CT, Stewart M, Kiehart DP (1990) Complete sequence of the Drosophila nonmuscle myosin heavy chain transcript: conserved sequences in the myosin tail and diffm'ential splicing in the 5' untranslated sequence. Proc Natl Acad $ci USA 87, 6316-6320 24 Kiehart DP, Ketchum A, Young P, Lutz D, Alfenito MR, Chang XJ, Awobuluyi M, Pesacretat TC, Inou6 S, Stewart CT, Chen TL (1990) Contractile proteins in Drosophila development. Annu N Y Acnd Sci 582, 233-251 25 Lengyel JA (1986) Blastoderm-specific genes in Drosophi. la. In: MolecularEntomology (Law J, ed) Alan R 1Ass,New. York, 223-231 26 Mabuchi I (1990) Cleavage furrow formation and actinmodulating proteins. Annu NYAcad $ci 582, 131-146 27 Mahowald AP, Kambysellis MP (1980) Oogenesis. In: The genetics and biology of Drosophila (Ashburner M, Wright TRF, eds) Academic Press, London, 141-224 28 Merrill PT, Sweeton D, Wieschans E (1988) Requirements for autosomal gem activity during precellular stages of Drosophila melanogaster. Development 104, 495-510 29 MillerKG, Field CM, Alberts BM (1989) AcUn.binding proteins from Drosophila embryos: a complex network of interacting proteins detected by F-actln affinity chromatography. J Cell Biol 109, 2963-2975 30 Nlisslein.Volhard C, Wieschaus E, Klucling H (1984) Mutations affecting the pattern of the larval cuticle in Drosophi. la melano&uster I. Zygotic loci on the second chromosome. Roux's Arch Dev Blol 193, 267-282 31 Pawson T, Bernstein A (1990) Receptor tyrosine kinases: genetic evidence for their role in Drosophila and mouse development. Trends Genet 6, 350-356 32 Pollard TD, Satterwhite L, Cisek L, Corden J, Sato M (1990) Actin and myosin biochemistry in relation to cytokinesis. Anna N Y Acud Sci 582, 120-130 33 Poodry CA (1990) shibire, a neurogenic mutant of Drosophi. la. Din, Biol 138, 464-472 34 Rappaport R (1971) Cytokinesis in animal cells, lnt Rev Cytol 31, 169-213 35 Rappaport R, Conrad GW (1963) Experimental analysis of unilateral cleavage in invertebrate eggs. J Exp Zool 176, 99-112 36 Rice TB, Garen A (1975) Localized defects of blastoderm formation in maternal effect mutants of Drosophila. Dev Biol 43, 277-286 37 Rubinstein PA (1990) The functional importance of multiple actin isoforms. BioEssays 12, 305-315 38 Sanders EJ (1975) Aspects of furrow membrane formation in the cleaving. Drosophila embryo. Cell Tiss Res 156, 463-474
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39 Schroeder TIE (1975) Dynamics of the contractile ring. In: Molecules and ceile movement (Inom~ S, Stephens RE, eds) Raven Press, 305-334 40 Schroeder TIE (1990) The contractile ring and furrowing in dividing cells. Ann N Y Acad Sci 582, 78-87 41 Schfipbach T, Wieschans E (1986) Maternal-effect mutations altering the anterior-posterior pattern of the Drosophila embryo. Roux's Arch Dev Biol 195, 302-317 42 Schfipbach T, Wieschans E (1989) Female-sterile mutations on the second chromosome of Drosophila melanogaster. I Maternal effect mutations. Genetics 121, 101-117 43 SchweisguthF, Lepesant JA, Vincent A (1990) The serendipity alpha gene encodes a membrane-associated protein required for the cellularization of the Drosophila embryo. Genes Dev 4, 922-931 44 SchweisguthF, Yanicostas C, Payre F, Lepesant JA, Vincent A (1989) c/s-regulatory elements of the Drosophila blastoderm-specific serendipity alpha gene: ectopic activation in the embryonic PNS promoted by the deletion of an upstream region. Dev Biol 136, 181-193 45 Siegfried E, Ambrosio L, Perrimon N (1990) Serine/threonine protein kinases in Drosophila. Trends Oenet 6, 357-362 46 Simpson L, Wieschaus E 0990) Zygotic activity of the nullo locus is required to stab'dize the actin-myosin network during cellulari,.~tlon in Drosophila. Development 110, 851-863 47 Sprenger F, Stevens LM, Nfisslein-Yolhard C (1989) The Drosophila gene torso encodes a putative receptor tyrosine idnase. Nature 338, 478-483 48 Sullivan W, Minden JS, Alberts BM (1990)daughterle~-abolike, a Drosophila maternal-effect mutation that exhibits abnormal centrosome separation during the late blastoderm divisions. Development 110, 311-323 49 Swanson MM, Pooclry CA (1981} The shibirets mutant of Drosophila: a probe for the study of embryonic development. Dev Biol 84, 465-470 50 Turner FT, Mahowald AP (1976) Scanning electron microscopy of Drosophila emhryogenesis. Dev 8iol 50, 95-108 51 Vincent A, Colot HV, RosbashM (1985)Sequenceand structure of the serendipity locus of Drosophila melanogoster. A denselytranscribed region including a blastoderm-six~ific gene. J Mol Blol 186, M9-166 52 Vincent A, O'Connell P, Gray MR, Rosbash M (1984) Drosophila maternaland embryo mRNAs transcribedfrom a single transcription unit use alternate combinations of exons. EMBO J 3, 1003-1013 $3 Warn RM, Mngrath R, Webb S (1984) Distribution of F-actin during cleavage of the Drosophila ~yncytial blastoderm. J Cell Biol 98, 156-162 $4 Warn RM, Robert-Nicoud M (1990) F-actin organization during the cellularization of the Drosophila embryo as revealed with a confocal laser scanning microscope. J Cell $ci 96, 35-42 55 Warn RM, Warn A, Planques V, Robert-Nicoud M 0990) Contractileproteinsin Drosophila development.Ann Rm, N Y Acnd Sci 582, 222-232 56 White JG, Borisy GG (1983) On the mechanisms of cytokinesis in animals cells. J TLeor Biol 101,289-316 57 WieschausE, Niisslein-Volhard C, Jiirgens G (1984) Mutations affecting the pattern of the larval cuticle in Drosophila melanogoster il. Zygotic loci on the third chromosome. Roux's Arch Dev Biol 193, 283-295 58 WieschausE, Sweeton D (1988) Requirements for X-linked zygotic gene activity during cellularization of early Drosophila embryos. Development 104, 483-494 59 Young PE, Pesacreta TC, Kiehart DP (1990) Dynamic changes in the distribution of cytoplasmic myosin during Drosophila embryogenesis. Development 111, 1-14 60 Zalokar M, Audit C, Erk I (1975) Developmental defects of female-sterile mutants of Drosophila melanoguster. Dev Biol 47, 419-432 61 Zalokar M, Erk 1 (1976) Division and migration of nuclei during early embryogenesis of Drosophila melanoguster. J Microsc Biol Cell 25, 97-106
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© Elsevier,Paris
Review
Genetic analysis of the cellularization of the Drosophila embryo F r a n f o i s S c h w e i s g u t h ! . , A l a i n V i n c e n t 2, J e a n - A n t o i n e L e p e s a n t a** zInstitut Jacques-Monod, CNRS et Um'versitd Paris 7, 2, place Jussieu, 75251 Paris Cedex 05; 2 Centre de Recherche de Biochimie et de Gdndtique Cellulaires. CNRS. 118, route de Narbonne, 31062 Toulouse Cedex, France (Received3 January 1991; accepted 7 May 1991)
Summary - The synchronous ceHularizationof the Drosophila embryo at the blastoderm stage provides a unique system for studying the molecular mechanisms involved in cytokinesis, using genetical and biochemical approaches. The cellularization process requires the major components of the embryonic cytoskeleton that are deposited into the egg during ongenesis. Genetical analysis indicates that it requires also the products of additional maternally-acting genes, as well as that of a limited set of zygotically-actinggenes. The cellularization defective phenotypes associated with small deficiencies uncovering these latter loci reveal specific steps within this complex process. The molecular analysis of these genes will ultimately provide meaningul insights into the normal process of cellularization. Among them, the serendipity a gene encodes a membrane-associated protein, which is exclusivelyacculumated during cellularization, and is required for the reorganization of the microfilaments as the onset of cellularization. Drosophila I cdlulm'inflon I cyllokJnesisI i¢tin I cytoskdetonI bl~oderm I s~rend/p/O,a
Introduction In animal cells, the cleavage of one cell into two cells at the end of mitosis, or cytokinesis, proceeds by constriction of the mother cell. Many decades of experimental research have provided different conceptual frameworks to account for the localized formation during telophase of the cleavage furrow and for its subsequent inward progression [34, $6]. It is now widely accepted that the mitotic apparatus somehow determines the furrow's position by inducing regional alteration of surface properties [34]. Thereafter cleavage activity itself relies upon a transient dynamic structure, the contractile ring, first described by Schroeder [39] as a belt of aligned microfllaments. Although the ultrastructural elements essential for cytokinesis have been identified long ago, the molecular mechanisms that regulate cytokinesis are still largely unknown (reviewed in [40]). In spite of great progress in the biochemical identification of the factors that modulate contractile proteins activity [26, 32], the mechanisms that control formarion, localization, maintenance and activity of the contractile ring during furrowing have not yet been determined. A molecular genetics approach might prove useful to evaluate the roles of the major cytoskeleton components, as well as to identify new molecules important for its regulation. Among the organisms with elaborate genetics, the fruit fly shows a striking form of cytokinesis early in development that consists of the cellularization of the syncytial embryo. A network of multiple cleavage furrows concertedly pushes into the syncytial Drosophila embryo to form simultaneously a monolayer of about 6 000 epithelial-like cells. The aim of this review is to summarize our current knowledge of the mechanisms involved in the cellulariza-
* Presentaddress:Departmentof Biology,B-022, Universityof California, San Diego, CA 92093 La JoHa, USA ** Correspondenceand reprints
tion process, and to emphasize more particularly the importance of molecular genetics in the analysis of this process. Cdlulsflzsflon of the wild.type Drosophila embryo The early Drosophila embryo initially develops as a syncytium. The first 13 mitotic divisions occur synchronously in the egg cytoplasm without cytokine#is [10]. The nuclei are initially located in the center of the egg. The majority of the nuclei migrate stepwiseto the cortical cytoplasm during cycles 7-10. Only 26 nuclei out of a total of 128 remain located in the center of the egg. These "viteilophag.e" nuclei stop dividing, become polypoid and never give me to cells. The 10-12 nuclei that reach the posterior pole of the embryo are incorporated into polar buds at cycle 9. These polar buds pinch off at the end of mitotic cycle 10 (80 man after fertilization). This process leads to the formation of the first cells, the pole cells which are precursors of the germ Hne cells. The rest of the nuclei are the somatic nuclei. They distribute as a uniform monolayer at the cortex of the egg and undergo three more nuclear divisions (cycles 11 to 13) to form eventually a cortical layer of about 6000 somatic nuclei. Each nucleus is surrounded by its own individualized cytoplasmic domain despite the fact that it is localized in a syncytium [20]. During each interphase of the mitotic cycles 11-13, this cytoplasmic domain forms a cap over the nucleus, which appears as a bulge at the surface of the embryo [58]. Early in metaphase, each cap extends and spfits into two daughter caps prior to nuclear division |53]. A membrane invagination appears around each chvlding nucleus at metaphase. It regresses during telophase while the two caps associated with the daughter nuclei reft',Tm, so that the cleavage furrow formed during nuclear ~:y:~;!e13 regresses prior to cellularization. These membrarie iiwaginations are referred to as transient cleavage furrows []3]. Cellularization occurs simultanz~,~u;ly across the whole
16
F Schweisguth et at
surface of the egg at the end of mitosis 13 and proceeds during the interphase of cycle 14 Coetwcvn 120 to 170 rain after fertilization) [10]. The plasma membrane invaginates down from the overlying surface between adjacent nuclei (fig 1). The invaginating membranes form an hexagonal array of cleavage furrows as viewed from above. All the cleavage furrows advance synchronously into the cortical cytoplasm, first at a slow rate during the so-called "slow phase" (between 120 and 155 min), and then more rapidly during the next 15 min of the "fast phase" of cellularization [58]. At the base of the cleavage furrow, the apposed membranes separate to form the furrow canal [12]. This structure appears as a triangular pocket on transverse sections, and as a circle on tangential view. The presence of the furrow canal distinguishes the cellularization furrow from the transient cleavage furrow that forms during each interphase of the nuclear cycles 11-13. The origin of the membranes that contribute to the increase in the plasma membrane surface remains unclear. Formation of multiple villous projections is observed prior to cellularization [12, 50]. These plasma membrane folds persist during the "slow phase" of cellularization. Fusion of newly synthesized endomembranes may contribute rather to the formation of the lateral membranes during this first phase [38]. The apical surface only flattens at the onset of the "fast phase", as if the plasma membrane accumulated in the villous projections had been pulled down in the cleavage furrow. It is supposed that this flattening process provides about half of the membrane surface required to enclose the blastoderm somatic cells [12]. Therefore, the difference in the rate of membrane inward progression between the slow and fast phases of cellularization may in part reflect the different mechanisms of membrane formation [12].
.. .
:
..
.
Cycle 13, telophase
Cycle 14, early phase I
Cycle 14, mid-cellulartzatlon
Cycle 14o late phase II
Fig 1. Schematic representation of the cellularization of the
D melanosasterembryo. Transverse views of an embryo from
telophase of mitotic cycle 13 until the end of cellularization. Nuclei are darkened. Stipplingindicates Foactindistribution. Centrosomes and microtubules are line.drawn. Stages are indicated below each view.
The Drosophila contractile ring network Accumulation of electron-dense filaments has been observed beneath the furrow canal during cellularization, forming a ring-like structure [12, 21]. This material progressively accumulates in a depressed zone of the surface plasma membrane [12, 21]. This circular depressed zone appears at the end of the telophase of the 13th syncytial division. Its diameter is defined by the midline of the underlying mitotic apparatus and the position where the cycle 13 transient cleavage furrow was previously located [12, 21]. This fibriUar material is composed of abundant filaments of actin [54], which are bidirectionally arranged [21], and of myosin II [59]. F-actin is found associated both with the lateral membranes, where it forms a roughly hexagonal network, and with the apical membrane folds during the first phase of cellularization. Small F-actin aggregates are also present within the cell below the cellularization front [54]. F-actin is no longer detectable in the cap region after the apical plasma membrane has flattened during the fast phase [54]. A number of actinbinding proteins are also specifically associated with the filamentous ring-like structure described above, or with other distinct subsets of actin filaments [29]. During membrane invagination, this ring-like structure remains associated with the furrow canal. Direct contacts between adjacent rings are commonly observed [54]. These interconnections hold all the rings together in a continuous network. The ring's diameter remains roughly constant during the slow phase of cellularization. It reduces in size during the fast phase of cellularization, so that each ring appears to be separate [54, 59]. Contraction of the ring pulls the membrane of the canal, which considerably enlarges, and results in the formation of the basal membrane of the epithelial.like blastoderm cells. After cellularization, a small bridge, which connects the cytoplasm of each cell with the yolk, persists until germ-band elonga. tion. The ring-like structure completely disappears at the end of cellularization. Local injections of cytochalasin [8, 10, 61], which depolymerizes F.actin, or of anti-myosin antibodies [24] block both the formation of this ring-like structure and the cellularization near the point of injection. These results indicate that the intactness of the ring-like structure described above is required for cellularization. The term "contractile ring network" has been proposed to describe this integrated array of ring-like structures in the Drosophila embryo [55], because they appear similar in composition, structure and function to the contractile rings of dividing cells as discussed below. The cellularization process is common to all insect species. It generally follows the description given above for D melanogaster. However many apterygote eggs, such as the Collembola eggs, do not develop as a syncitium, since early nuclear divisions are followed by complete cytokinesis [17]. Variations have also been reported in certain hemimetabolous insects. In lsoptera and Cheleutoptera, cortical nuclei distribution is highly polarized in the differentiating syncytial embryo. The nuclei are closely packed near the posterior pole, while being widespread elsewhere. By contrast to the Drosophila uniform cellular blastoderm, which is formed by the synchronous cellularization of a monolayer of evenly distributed nuclei, the differentiated cellular blastoderm is formed by the progressive aggregation of cells from the anterioly located extraembryonic region to the embryonic primordium, the latter resulting from the localized cellularization of the nuclei
Cellularizationof the Drosophila embryo clustered near the posterior pole [3]. The presumed differences between the invagination of the plasma membrane in the extra-embryonic versus the embryonic primordium has not been analyzed in these species. It would be of interest to analyze the formation of the contractile network at the borders of the embryonic primordium in these species. Functional similarities between the contractile ring network of the Drosophila embryo and the contractile ring of dividing cells The contractile ring in dividing animal cells is mainly composed of actin and myosin filaments, arranged bidirectionally in a transient ring structure. It apparently forms primarily through the reorganization of pre-existing cortical actin filaments that directionally move towards the equator region of the dividing cell [5, 6]. Sliding interactions between actin and myosin f'daments are generally thought to provide the furrowing force [40]. Indeed, injection of cytochalasin, or of anti-myosin II antibodies, in dividing cells results in the disruption of the contractile ring and in the blockage of the inward progression of the cleavage furrow (see [40] and references therein). Likewise, deletion of the single myosin II gene in Dictyostelium has provided clear evidence that myosin II is an indispensable motor for cytokinesis [11]. The Drosophila contractile ring network therefore appears highly similar in composition and structure. However, there are no clear data relevant to the mechanism of assembly of actin and myosin filaments into a contractile ring network. It is not known whether de novo assembly of unpolymerized subunits onto nucleation sites accounts for the contractile ring network formation, or whether conical pre-existing fdaments migrate from the cap towards the depressed zone where the contractile rings appear [21], as described for cytokinesis [$, 6, 21]. It has been suggested from the description of the F-actin organization by fluorescence staining that the actin filaments stocked in cytoplasmic aggregates become progessively part of the contractile rings [54]. This hypothesis is consistent with the mechanisms proposed for contractile ring formation in dividing cells by Cao and Wang [5, 6]. Likewise it is not yet clear whether actin and myosin filaments provide the furrowing force during cellularization in a manner similar to cytokinesis, or whether they are required to stabilize the network across the whole surface of the egg during cellularization. The results obtained by injections of cytochalasin and of anti-myosin II antibodies, ie local inhibition of cellularization, are consistent with the hypotheses that the contracting ring network acts as a motor for cellularization or as a structural passive element. The structural similarities presented above between contractile rings involved in cellularization and cytokinesis argue in favor of the assumption that the contractile ring network plays a motor role. These similarities appear even more striking if one considers the unilateral, or heartshaped, cleavage described in coelenterate eggs [35]. In this variant of cytokinesis, the mitotic apparatus is excentrically located at the animal pole. The furrow only appears in the animal pole surface, and proceeds unilaterally towards the vegetal pole. In the Drosophila embryo also, the mitotic apparatus is localized in the cortic',d cap, close to the apical surface, which by analogy corresponds to the animal pole surface. The yolk-rich center of the egg would be then considered analogous to the vegetal pole of the
17
coelenterate egg. However, these two cleavage figures still differ by the relative orientation between the cleavage furrows and the contractile rings. The contractile ring is located in the same plane as the advancing cleavage furrows during cytokinesis, even in the case of the unilateral cleavage of coelenterate egg [40], while the Drosophila contractile ring network is oriented perpendicularly to the invaginating furrow membranes. This difference in geometrical arrangement indicates that the role of the contractile ring network during cellularization cannot be directly inferred from the one proposed for the contractile ring in dividing animal cells. The contractile activity of the F-actin meshwork might be required only at the end of cellularization to pinch off cells. During the fast phase of cellularization, each contractile ring becomes progressively reduced in size as the cleavage furrows approach the inner yolk mass [54]. Sliding interactions between actin and myosin filaments are thought to be responsible for this reduction in size, which results in furrow canal enlargement and basal membrane formation [54]. Because contraction of each ring correlates with the formation of the basal membranes proceeding in the same plane as the one defined by the contractile ring network, it has been suggested that additional forces may be required to either "pull" or "push" down the cleavage furrow in a direction orthogonal to the one defined by the contractile ring [55]. The contractile ring network might then be essentially required during the first slow phase of cellularization to ensure the cohesiveness of the entire matrix as a structural or tension bearing element. These speculative considerations should make it clear that the mechanism of cellularization is far from being understood. The phenotypical analysis of cellularization defective mutations may help us to dissect the discrete mechanisms of this process. Phenotypes of cellulaflzatlon mutant embryos Mutations that affect cellularization have been recovered in genetic screens for female-sterile loci [36, 42, 60], and for zygotically required loci [28, 58]. Cellularization of the syncitial blastoderm embryo thus depends upon both maternal and zygotic factors. Indeed, most gene products required for early embryogenesis are synthesized during oogenesis and deposited into the egg [27]. In addition, specific genes are zygotically transcribed prior to cellularization, as early as nuclear cycle 11 [9, 44], thereby accounting for the zygotic contribution. T:le phenotypes associated with these two classes of mutations are detailed in the following paragraphs. Maternal-effect mutations
About 25 maternal-effect nmtations have been isolated that affect the cellularization process [36, 41, 42, 60]. In most cases, however, their cellularization phenotype has not been yet analysed in great detail. Thefs(l)1459 ~ mutation is among the best described [60]. At high temperature, the development of embryos laid by females homozygous for thefs(l)1459 ts mutation is specifically arrested at the cellular blastoderm stage. The thermosensitive period of this mutation is restricted to this developmental stage. Electron micrographs show that cell membranes do not invaginate in fs(1)1459 ts embryos at the time when the nuclei start to elongate. The furrows remain short and irregular. No furrow canals can be detected at the base of
18
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the membrane furrows. The nuclei eventually migrate deep into the periplasm. Interestingly, the fs(1)1459 ts embryos are in many aspects similar to cytochalasin injected embryce, suggesting a defect in microfdament-based activity. As a general rule, the cellularization defects associated with zygotic or maternal-effect mutations are distributed uniformly across the whole surface of the embryo. A few maternal-effect mutations (mat(3)3 and mat(3)6 [36]; torso and trunk [41]; maternal effect of I(l)pole hole [11]) exhibit |ocaliT~i defects and constitute notable exceptions. The mat(S)3 mutation results in a failure of cell formation on the dorsal-posterior region. Conversely, in matO)6 mutant embryos, cellularization is detectable only at poles where complete cells form [36]. In both mutants the noncellularized region presents extensive defects: the nuclei move away from their apical position in the cortical cytoplasm, which is itself invaded by yolk. The initial defects detectable in these mutant embryos have not yet been analysed in detail. Mutations in the torso, trunk and I(l)pole hole genes, which primarily affect abdominal pattern formation, are three other interesting exceptions. Loss of function alleles of these genes shows localized cellularizatinn defects at the posterior pole [1, 41]. The torso gene activity is specifically restricted to the posterior pole [7]. The torso gene encodes a membrane receptor with a tyrosine kinase cytoplasmic domain [47]. The torso protein is thought to regulate the activity of ser/thr kinases involved in the induction of posterior pole development [2, 31]. One of these kinases is encoded by the l(l)pole hole gene [1, 45]. The observation of localized defects, and the fact that cellularization appears to be a uniform process across the whole surface of the embryo, raise an apparent contradiction. In order to reconcile these contradictory terms, we propose that protein phosphorylation is a key control mechanism of cellularization across the whole surface of the embryo, and that certain mutations affecting a phos. phorylation cascade in a specific region of the embryo are able to interfere with the ceilularization process in that same area. Then, the fact, that cellulariLation defects are detectable in a specific region of the embryo surface does not necessarily imply that the mechanism of cellularization is not identical in all somatic cells. It remains to be determined whether the localized defects observed in mat(S)3, mat(S)6 and trunk embryos are also secondary consequences of an altered process of pattern formation. Zygotically required loci
The Drosophila genome has been essentially saturated with zygotic lethal mutations, and those resulting in specific cuticular phenotypes have been genetically analysed [18, 30, 57]. None of the zygotic lethal mutations isolated in the course of this extensive screen resulted in a cellularization phenotype. It was however already known that synthesis of zygotic factors is required for cellularization, as shown by inhibiting de novo transcription through injection of ~-amanitin in cleave~e-stageembryos, which results in abnormal cellularization [4]. One possible explanation for the non-isolation of zygotic lethal mutations affect. ing cellularization is that no cellularization defective mutants exhibited a reproducible cuticular phenotype, and the putative cellularization mutations were therefore discarded by these authors [18, 30, 57]. Another possibility is that, among the putative zygotic cellularization mutations, those which result in the formation of an abnormal number of cells do not result in embryonic lethality. Indeed, embryos
laid by females homozygous for the daughterless-abo-like (dal) maternal-effect semi-lethal mutation show an impressive ability to recover from a secondary cellularization defect [48]. These embryos cellularize with only about onehalf the normal number of cells, and 8~/e even develop to adulthood [48]. These remarks suggest that detection of cellularization phenotypes may require the direct observation of blastoderm-stage embryos. Therefore, the development of living embryos deficient for cytologically defined portions of the genome has been followed in order to identify genomic regions that are zygotically required for the cellularization of the embryo [28, 58]. Attached X-chromosomes, compound autosomes and Y translocations were used to generate these deficient embryos. After scanning the whole genome, only six chromosomal regions were identified that were associated with a unique cellularization phenotype when deleted. It can thus be predicted that each of these genomic regions contains at least one zygotic cellularization locus. In all cases, numerous other defects appear after cellul"anzation in these deficient embryos which finally die. For this reason, the phenotype associated with each deficiency, as detailed below and summarized in table I, corresponds only to the earliest detectable one. The establishment of the hexagonal F-actin network requires gene activity from three distinct genomic regions. Deficiencies encompassing these regions define the loci 6F-TA or hullo, 99D and 100 AC, so-named after their respective position on the cytological map of the polytene chromosomes. The phenotypes associated with the lack of hullo, 99D or 100AC gene activities are initially s!m_i!ar, with hullo defects being the most severe. The phenotype associated with 100AC deficiencies differs from the two others later in cellularization (see below). These pheno. types consist in erratic disruptions of the cytcekeleton early at mitotic cycle 14 (fig 2) leading to the formation of cells that contain multiple nuclei [28, 43, 46, $8]. These phenotypes suggest that the reorganization of the actin-myosin filaments into an hexagonal network prior to membrane invagination is regulated, but do not indicate whether this reorganization proceeds through directional migration of pre existing filaments or de novo filament polymerization at localized nucleation sites. It should also be noted that no specific abnormalities are detected at the syncytial blastoderm stage. Specifically, the transient cleavage furrows normally form at the interphases of mitotic cycles 11-13. This indicates that the formation of pseudocleavnge furrows during cycles 11-13 and of cleavage furrows during cellularization are partly under separate genetic control. The lack of 100AC p n e activity also results in deregulated contractile activity of the contractile ring network. Each contractile ring reduces in size before midcellularization, as the actin and myosin f'daments progressively contract, entrapping the nuclei at the cellularization furrow [28]. Although the cytoskeleton remains firmly anchored to the plasma membrane, the invagination of the latter is inhibited [28]. This phenotype suggests that the 100AC locus is required either to keep the contractile rings interconnected in an integrated network, or to regulate temporally the sliding interactions between actin and myosin filaments to prevent each ring from contracting before mid-cellularization. The plasma membrane invagination requires two other loci, named 26BF and 40AC. The analysis of their respective phenotype provides a functional distinction between the slow and fast phases of cellularization [28]. These two
Cellularizationof the Drosophila embryo
19
Table I. Summary of the phenotypes associated with the zygotically-required loci. For each locus listed in the leftmost column, the corresponding ceHularization-defective phenotype is diagrammed. Plasma membrane is drawn as a line and nuclei are darkened. The arrow indicates the invaginafion rate of the plasma membrane, the thinner part corresponding to the initial slow rate, the thicker part corresponding to the following fast rate.
Locus
Chromosomal localization
References
nullo
6F-TA
[46, 58]
sly ~
99D4-8
[28, 43l
IOOAC
IOOAC
[28]
Formation of plurinucleate ceils and premature closure of contractile rings
26BF
26BF
[281
No membrane invagination during the slow phase
40AC
40AC
[281
No accelerated rate of membrane invagination during the fast phase
[28]
Disruptions in the conical localization of the somatic nuclei at an early stage of cellularization
71C-75C
71C-75C
Phenotype
Formation of plurinucleate cells
phases, initially defined by their different rates of membrane lnvngination [$8], can also be distinguished by their distinct genetic requirements. Deletion of the 26BF locus results in a lack of membrane invngination during a 40 rain period after completion of mitosis 13, a period during which the slow phase of cellularization normally takes place. Thereafter, the plasma membrane rapidly invaginates, at a rate similar to that of the fast phase of cellularization in wild-type embryo [28]. Cellularization is not uniformly completed in these embryos before gastndation. Conversely in the absence of 40AC gene activity, no fast phase is detectable. The plasma membrane continues to invnginate at a slow rate until gastrulation, which takes place at the correct time [28]. This clearly indicates that cellularization is a two-step process, and that the two phases are in part functionally independent. Finally, deletion of the locus 71-75C affects the apicalbasal distribution of the nuclei, as many nuclei sink into the interior of the embryo [28]. A s~dlar defect is observed following cytochalasin B injection [8] that results in the extensive reorsanization of both microfilaments and microtubules. In contrast to cytochalasin injected embryos, membrane invagination proceeds normally in embryos homozygous for a 71C-75C deficiency. This difference suggests that these two actin-based processes, ie the cortical localization of the nuclei and the invagination of the
plasma membrane, involve distinct specific factors, one of which is encoded by the locus 71C-75C. No specific mutation in any of these six loci has yet been Lqolated. Whether a mutation leading to the complete lack of a function in any one of these six zygotic loci is lethal to the embryo, or affects other morphogenetic processes is therefore not yet known. Related loci
There exist additional mutations that affect in a nonspecific manner the cellularization process. The shibire thermo-sensitive mutation (shits) is one well-documented example. In shP homozygous animals, endocytosis is inhibited at restrictive temperature. This inhibition in turn results in various developmental defects [33], including cellularization defects. When cellularizing blastoderm embryos are shifted at restrictive temperature, the furrow membranes are disrupted into numerous vesicles [49]. This suggests that shibire activity i~ required for a general function related to cycling of vesicles and/or to membrane stability. The last group of mutations, whose phenotypes are possibly related to cellularization, corresponds to cytokinesis defective mutations. Two such mutations, l(l)d degll and
20
F Schweisguthet al
/ /
B
Fig 2. Cellularization defects associated with the 99D deficiency, Embryos homozygous for the deficiency 99D (Df(3R)X3F: [43]) (B, D and F) are compared to wild.type embryos (A, C and E). A and B. Sectionsthrough surface cortex reveal the irregular distribution of the nuclei in deficient embryos. C-F. Disruptions of the F-actin array, visualized on surface views (C and D) and side views (E and F) of whole-mount embryos using fiuorescently labelled phalloidin. Bars: 5 pro.
1(3) 7m.62, have been recognized among a collection of
larval and/or pupal lethal mutations as the ones producing highly polypoid cells in larval brain squashes [15]. The 1(33 7m-62 locus, or spaghetti-squash, has recently been shown to encode the myosin [I regulatory light chain [19]. It is not yet known whether these two loci are required for ceilularization.
The cellularization defective 99D locus is the blastodermspecific serendipity ~ gene The isolation of the maternal-effect mutations and the cytological mapping of the zygotically required loci provide the first steps for going beyond the description of the cytoskeletal components and moving on to the molecular identification of these factors.
The smallest deficiency that defines the 99D locus covers 9 cytological bands, and encompasses many transcription units [52], among which is the serendipity ~ (sty ~) gene [51], located at position 99D4-8. Transcription of the sty gene starts at nuclear cycle 1I, peaks at the onset of cellularization and rapidly decreases during the fast phase of cellularization ([44] and F Schweisguth, unpublished results). Sequence analysis of the encoded protein does not reveal any significant homology to known proteins [51]. Since the zygotic ceilularization phenotype associated with 99D deficiencies [28] appears at the onset of zygotic transcription, it probably results from the non-expression of a strictly zygotic and early expressed gene. The hypothesis that the cellularization phenotype associated with deficiencies encompassing the 99D region is due to the lack of sty ~ gene activity was tested by P element rescue experiments. Stable insertion of a functional sty ~ gene into
Cellularization of the Drosophila embryo the genome of a 99D deficient embryo is able to restore a normal cellularization process. The sty ~ gene activity alone is therefore sufficient to rescue the cellularization defects associated with the 99D deficiency, indicating that the sty • gene is required for cellularization [43]. The sty gene is the first gene to be identified that is specifically required for the cellularization of the Drosophila embryo. None of the other blastoderm-specific genes, isolated by differenti~ cDNA screening [25], is located on the polytene chromosomes map within intervals that define the cellularization defective zygotic loci. Using specific antibodies, the 58 kDa sry ~ protein has been detected in association with the folded apical membrane at the onset of cellularization, and with the furrow membranes during invagination. The sry ~ protein is specifically accumulated at the level of the furrow canal during cleavage formation [43], and appears to be part of the contractile ring (fig 3). This localization is similar to the ones given for the actin and myosin filaments [54, 59]. No sry ~ protein or transcripts are detectable after midgastrulation. During the whole fife-cycle of the fly, the sry protein accumulates for only one hour, indicating that normal cytokinesis does not require the presence of the sry ~ protein. The sry ot protein is also dispensable for pole cell formation at mitotic cycle 10. The phenotype of the 99D deficient embryos (fig 2) suggests that the sry a protein is required for the initial reorganization of the apical microfilaments into a regular hexagonal network. One simple hypothesis would then be that the sry ~ protein binds to F-actin. However, it is not among the 40 actin-binding proteins purified from early embryos by F-actin affinity chromatography ([29] and
21
C Field, personal communication), and no putative actinbinding motifs have been identified within the predicted sequence of the protein. Conclusions and prospects The genetic analysis of cellularization is still at an early stage. In most cases, the maternal-effect mutations still await an accurate phenotyr'." description. With regards to the zygotically-required loci, no specific mutations, as opposed to deficiencies, have yet been recovered. Nevertheless several conclusions may be drawn already. First, one of the most remarkable features of the phenotypes described above is their specificity for particular aspects of cellularization. Such specifities in phenotypes should make it possible to dissect the discrete mechanisms of this complex cellular process. This appears especially true for the zygotically-required loci. This could be simply due to a more detailed phenotypic ~atysis of the zygotic loci as compared to the maternal ones. Alternatively, one attractive possibility is that the zygotically-acting genes encode regulatory factors whose accumulation must be developmentally regulated, while maternally-acting genes mostly contribute to the accumulation of general factors, such as the major and ubiquitous components of the cytoskeleton. Also, none of these mutations specifically affect the timing of cellularization. Cellularization is never observed prior to cycle 14. The onset of cellnlarization does not depend upon nuclear density. As described above, ceilularization occurs normally at mitotic cycles 14 in dal- embryos,
Fig 3. Localization of the sty, protein in the contractile ring. Top view of the contractile ring network of a wild type mid-cellularizing embryo of D melanogasterrevealing the distribution of the sry • protein detected by indirect immunofluorescence using immunopurifled anti-sry • antibodies and rhodamine-conjugated secondary antibodies excited at 488 nm. This photograph was obtained using the × 40 objective on a Biorad MRC 100 confocal laser scanning microscope. Bar: 5 ttm.
22
F Schweisguthet al
though there is only one-half the normal number of nuclei [48]. The timing of cellularization is also not tightly coupled to the transition between synchronous nuclear- cycles and spatio-temporally regulated mitosis. Indeed, cellularization be~i'ns at the proper developmental time even when an extra round of syncytial division is observed, as, for example, in maternal haploid embryos [9]. The maternal haploid mutation, originally named fs(1)572, was isolated as a maternal-effect mutation affecting cellularization by Zalokar et al [60]. In these embryos the 14th synchronous mitosis interrupts cellularization, causing developing cell membranes to partially regress. The mechanism that determines the onset of cellularization at the end of nuclear cycle 13, in a manner independent of the nuclear density and of the total number of synchronous syncytial cycles, remains unknown. Surprisingly, none of the ceilularization defective mutations isolated to date correspond to the single myosin II gene [23], or to any member of the cytoplasmic actin encoding mnltigene family [13, 14]. Two very different reasons may account for this observation. In the case of the myosin II gene, strong loss of function mutations would presumably results in cell autonomous lethality. This means that such mutations would surely affect cell vialibity during oogenesis, therebey preventing the production of fertilized eggs. This cell lethal effect might be expected to occur in germ line clones homozygous for such mutations, generated in heterozygous mothers. The problem differs in nature for the actin genes because the coding sequences of the two cytoplasmic actin genes are highly homologous [14]. Therefore a mutation in one of the two cytoplasmic actin genes might be compensated for by the other unmurated one, or by any one of the four other actin genes. This hypothesis assumes that none of the isoforms serves a highly specialized and non-compensable function during cellularization (see [37] for a discussion of the differential iso-actin utilization in nonmuscle cells). The phenotypical evaluation of the function of the actin and myosin filaments during cellularization and cytokinesis in Drosophila embryo awaits the isolation of conditional mutations in the myosin II and actin genes, such as thermosensitive mutations. In the case of the actin genes, the desired mutation has to be not only conditional but also "dominant negative", so that mutated actin gene products interfere with the activity of the wild-type products, through the incorporation of inactive subunits in multimeric filaments. These types of mutational events are expected to be rare, and have indeed not yet been observed. Nevertheless, the ability to manipulate in vitro the cloned genes and to reintroduce them into the Drosophila genome opens the way to the design of conditional dominant negative mutations, as discussed by Herskowitz [16]. Finally, in almost all of the zygotic and maternal-effect mutant embryos described above, pole cells form normally. Pole cell and blastoderm cells formation appear to be under separate genetic control. The mechanism of pole cell formation has not been dealt with in this review. The combination of classical genetics, molecular biology and biochemistry makes the cellularization of the Drosophila embryo an attractive model for the elucidation of fundamental questions on cytokinesis. The mechanism that determines the nuclear cycle at which cellularization occurs, the nature of the factors that determine the position where cleavage furrows will subsequently form, the way the contractile ring network is generated and maintained, remain largely unknown. Many cellularization defective loci, but certainly not all, have
been isolated. In an independent approach, a number of actin-binding and microtubule-associated proteins have been identified by affinity chromatography [22, 29]. These two approaches may ultimately converge to define a limited set of genes. Molecular and biochemical studies will then be required for a better understanding of the function of these genes. Until now these data remain preliminary, and any attempt to propose a model for the cellularization of the Drosophila embryo would still largely rely on studies of cytokinesis in cultured cells or in marine invertebrate eggs. Hopefully, the genes that regulate ceilnlarization will be cloned in the near future, the proteins they encode characterized and their role during cellularization and cytokinesis evaluated in vivo by genetical means. The molecular dissection of the cellularization process may then greatly assist analysis at the molecular level of the universal regulatory mechanisms involved in cytokinesis. Acknowledgments We thank E Wieschaus and DP Kiehart for preprints, C Field for communicating unpublished results, J Deutsch and H Brock for comments, G C~raud for help with confocal microscopy and A Kropfinger for revision of the manuscript. References
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