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1863 Journal of Cell Science 107, 1863-1873 (1994) Printed in Great Britain © The Company of Biologists Limited 1994 The nullo protein is a component of the actin-myosin network that mediates cellularization in Drosophila melanogaster embryos Marya A. Postner and Eric F. Wieschaus* Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA *Author for correspondence SUMMARY After the 13th nuclear division cycle of Drosophila embryogenesis, cortical microfilaments are reorganized into a hexagonal network that drives the subsequent cellularization of the syncytial embryo. Zygotic transcription of the nullo and serendipity-α genes is required for normal structuring of the microfilament network. When either gene is deleted, the network assumes an irregular configuration leading to the formation of multinuceate cells. To investigate the role of these genes during cellularization, we have made monoclonal antibodies to both proteins. The nullo protein is present from cycle 13 through the end of cellularization. During cycle 13, it localizes between interphase actin caps and within metaphase furrows. In cellularizing embryos, nullo co-localizes with the actin-myosin network and invaginates along with the leading edge of the plasma membrane. The serendipity-α (sry-α) protein co-localizes with nullo protein to the hexagonal network but, unlike the nullo protein, it localizes to the sides rather than the vertices of each hexagon. Mutant embryos demonstrate that neither protein translationally regulates the other, but the localization of the sry-α protein to the hexagonal network is dependent upon nullo. INTRODUCTION taposition: the sides of each polygon in the array are formed by converting the ‘fuzzy’ actin organization at the cap margins into more finely aligned actin filaments (Simpson and Wieschaus, 1990). Each hexagonal interface of the actin network defines the site of membrane invagination. Cytoplasmic myosin co-localizes with actin in the hexagonal network (Warn et al., 1980; Young et al., 1991), and both filamentous actin and functional cytoplasmic myosin are required for membrane invagination (Zalokar and Erk, 1976; Foe and Alberts, 1983; Kiehart et al., 1990). Contraction of this actin/myosin array has been postulated to provide a mechanistic force driving the invagination. This role for the actinmyosin network is based in part on an analogy with the ‘contractile rings’ of actin and myosin that are thought to drive invagination of the plasma membrane during conventional cytokinesis (Mabuchi, 1986; Salmon, 1989; Schroeder, 1990; Satterwhite and Pollard, 1992). Most of the components of the hexagonal array are supplied by maternal transcription during oogenesis and are already present as RNA or protein in the unfertilized egg. Cellularization, however, marks the point in Drosophila development when the embryo becomes dependent on gene products supplied by the embryo’s own transcription (Arking and Parente, 1980; Edgar and Schubiger, 1986). A small number of genes have been identified whose zygotic products are required for the formation of a normal actin array (Wieschaus and Sweeton, 1988; Merrill et al., 1988). Embryos lacking either the nullo or the serendipity-alpha (sry-α) gene show very similar In Drosophila embryos, the early nuclear divisions are not followed by cytokinesis and the embryo initially develops as a syncytium. This organization persists until after the 13th division, at which time the embryo consists of approximately 6,000 nuclei arranged in a monolayer in the embryo’s cortex. Subdivision of the cortical cytoplasm into individual cells is known as cellularization. During this process, plasma membrane invaginates from the surface in a roughly hexagonal pattern, precisely separating each nucleus from its immediate neighbors. Once the membrane has reached a depth of about 25 µm, the base of the invaginating membrane furrow begins to widen, eventually separating the newly formed cells from the underlying yolk. The resulting cellular blastoderm consists of a single layer of columnar cells surrounding the central yolk sac. During the first ten minutes of cycle 14, a highly organized array of F-actin is formed on the cytoplasmic face of the invaginating plasma membrane (Fig. 1, see also Warn and Magrath, 1983; Simpson and Wieschaus, 1990; Warn and Robert-Nicoud, 1990; Schejter and Wieschaus, 1993). Prior to formation of the array, the cortical actin of the embryo is organized in ‘caps’ overlying each nucleus. Initially the caps formed in cycle 14 resemble those seen in the preceding interphases. However, in contrast to earlier caps, which remain static during interphase, the cycle 14 caps soon enlarge until their bases touch. The actin array arises in regions of cap jux- Key words: Drosophila embryo, cytokinesis, contractile ring 1864 M. A. Postner and E. F. Wieschaus abnormalities in the actin array: some of the sides of the hexagons are unusually thick while others are extremely thin or missing altogether (Fig. 1B,C). In nullo embryos, the initial few minutes of network formation appear normal (Wieschaus and Sweeton, 1988; Simpson and Wieschaus, 1990). However, at the onset of membrane invagination, network formation is incomplete and the uneven distributions and sporadic disruptions in the actin-myosin network become obvious. Once underway, network and membrane invagination appear to proceed normally: neither the kinetics of membrane extension nor the length of the newly formed cells is significantly different from that observed in wild-type embryos. However, cleavage furrows do not invaginate where the network is discontinuous and multinucleate cells form. The only obvious difference between nullo and sry-α mutant embryos is that sry-α embryos have fewer multinucleate cells (Merrill et al., 1988; Schweisguth et al., 1990; E. Schejter, personal communication). Molecular characterization of the nullo and sry-α genes has revealed that both genes encode single, blastoderm-specific transcripts that are uniformly distributed throughout the syncytial embryo and accumulate in large amounts over a short period of time (Vincent et al., 1985; James and Vincent, 1986; Rose and Wieschaus, 1992). Transcript levels reach a sharp peak around the onset of cellularization and subsequently decrease in a rapid, spatially patterned manner. The main difference in the transcription pattern of the two genes is that sryα transcripts arise, peak and decline slightly later than nullo. Neither gene is required for the transcription of the other (Rose and Wieschaus, 1992). The sry-α protein is 58 kDa in size, lacks extensive homology to any known proteins and shows few structural motifs (Ibnsouda et al., 1993). Immunolocalization indicated that during cellularization sry-α protein localizes to the leading edge of the invaginating plasma membrane (Schweisguth et al., 1990). Like its transcript, the sry-α protein is short-lived. The nullo gene is predicted to encode a 23 kDa protein lacking homology to known proteins, including the sry-α protein. Sequence analysis demonstrated that the nullo protein has an excess of basic amino acids (predicted pI is 11.4) and suggested that the protein may be myristoylated (Rose and Wieschaus, 1992). However, previous studies did not address intracellular localization of the nullo protein or its specific cell biological function during cellularization. While many broad questions regarding the mechanisms of cellularization remain unanswered, several specific questions about the nullo and sry-α proteins are amenable to experimentation: do the proteins directly interact with the cellularization machinery or are they indirect participants in cellularization? What are the functions of the nullo and sry-α proteins during the process? Is the similarity of their mutant phenotypes due to their participation in a common pathway or do the two proteins function independently? To address these questions, we have generated monoclonal antibodies to both nullo and sry-α proteins, and examined their distribution during cellularization in both wild-type and mutant embryos. MATERIALS AND METHODS Genotypes and stocks used Ore-R was used as the wild-type stock. Embryos with the nullo phenotype were collected from balanced stocks containing deficien- cies that uncover the nullo locus. The deficiencies Df(1)6F1-2 and Df(1)LIMDF were most commonly used (for description see Simpson and Wieschaus, 1990; Rose and Wieschaus, 1992). Embryos with the sry-α phenotype were collected from a stock that is heterozygous for Df(3R)X3F, which uncovers the sry-α gene. Since no point mutations exist for either gene, the mutant phenotype only arises in deficiency embryos. The terms ‘nullo mutant’ and ‘sry-α mutant’ are used to describe the deficiency embryos. Production and screening of monoclonal antibodies Monoclonal antibodies to the nullo protein were generated using a nullo-glutathione S-transferase fusion protein as the antigen. An inframe fusion of the entire nullo protein to the carboxyl terminus of glutathione S-transferase (Smith and Johnson, 1988) was constructed in the following manner. The nullo coding region was PCR amplified from the nullo M1 cDNA (Rose and Wieschaus, 1992) using primers homologous to the ends of the coding region. The primers also contained an external stretch of bases that lacked homology to nullo and contained an EcoRI restriction site. The resulting PCR product was digested with EcoRI and ligated into the EcoRI site of pGEX-2T (Pharmacia). The orientation of the inserts was determined by restriction mapping. The pGEX-2T-nullo plasmid was transformed into Escherichia coli strain JM101 and the production of fusion protein was induced with 1 mM IPTG (Pharmacia). After 3 hours, the cells were harvested. Because the fusion protein was stubbornly insoluble, it was excised from a preparative acrylamide gel, electroeluted with Elutrap (Schleicher and Schuell), and dialyzed against MTPBS (Smith and Johnson, 1988). The purified protein was used to immunize two mice and monoclonal antibodies were produced following standard protocols (Harlow and Lane, 1988). Supernatants from the monoclonal lines were tested by western blot for recognition of the fusion protein and of glutathione S-transferase. The 23 lines that recognized only the fusion protein were tested by western blot for reactivity with proteins from two- to three-hour Ore-R embryos. Monoclonal supernatants 5C3-12 and 2F8-18 specifically recognize the nullo proteins. Because the 5C3-12 antibody reacts more strongly with the nullo proteins than does 2F8-18, it was used preferentially unless otherwise indicated. Monoclonal antibodies to the sry-α protein were generated using a truncated version of the sry-α protein as the antigen. This protein, which contains amino acids 46 to 530 of sry-α, was produced from a T7 RNA polymerase-inducible vector (Studier and Moffat, 1986). The plasmid, pPαNN, contains a NarI to NcoI fragment of the sry-α gene cloned in pET3a. It was generously provided by Alain Vincent. The plasmid was transformed into E. coli strain BL21-Lys S. Fusion protein production was induced for three hours with 1 mM IPTG. The cells were lysed by freezing with 3.3 mg/ml lysozyme and then thawing. Inclusion bodies were purified by isolating all proteins insoluble in DOC buffer (200 mM NaCl, 1% sodium deoxycholate, 1% NP40, and 1 mM DTT). The protein pellet was washed three times with 0.5% Triton X-100, 1 mM EDTA and 1 mM DTT, before being resuspended in TE (10 mM Tris, 1 mM EDTA). This protein preparation was greatly enriched for the truncated sry-α protein. Two mice were immunized with it and monoclonal antibodies were produced. The monoclonal supernatants were screened by western blot to test reactivity with the truncated sry-α protein that served as the antigen. Reactive supernatants were further tested for reactivity with a 58 kDa protein from two- to three-hour wild-type embryos. Monoclonal supernatants 1G10, 3H6, 4G4, 6B12 and 6F4 all react strongly with the sry-α protein. Antibody 1G10 was used in most instances. Immunoprecipitation Two- to three-hour wild-type embryos were dechorionated, ground in 80 mM Tris with 2% SDS, and boiled for five minutes. The extracts were chilled on ice. Modified RIPA buffer (50 mM Tris, 300 mM NaCl, 1% NP40, 0.5% sodium deoxycholate) and 10% Triton X-100 were added to the extracts to achieve a final concentration of 0.2% nullo protein in Drosophila embryos 1865 SDS and 1% Triton X-100. Samples of 75 µl each of nullo monoclonal antibodies 5C3-12 and 2F8-18 were incubated with the protein extracts for one hour at 4˚C. Then, 50 µl of 50% Protein A-Sepharose beads (Pharmacia) in RIPA buffer (above recipe plus 1% SDS) were added and incubated at 4˚C. After one hour, the pellet was recovered and washed twice in RIPA buffer. Antibody staining of embryos Two- to three-hour embryos were dechorionated and fixed by one of two procedures: (1) fixation for 20 minutes with 18.5% formaldehyde saturated with heptane followed by manual devitellinization or (2) boiling for 10 seconds in Triton-salt solution (68.4 mM NaCl, 0.03% Triton X-100) followed by the addition of a vast excess of ice-cold Triton-salt solution, devitellinization using methanol and heptane, and post-fixation of at least one hour in methanol. The fixed and devitellinized embryos were incubated for one hour at room temperature with PBT10 (PBS with 10% BSA and 0.1% Tween-20). Incubations with the primary antibodies were performed overnight at 4˚C: sry-α monoclonal antibody 1G10 was diluted 1:25 in PBT1 (PBS with 1% BSA and 0.1% Tween-20); nullo monoclonal 5C3-12 was diluted 1:15 in PBT1; antisera to cytoplasmic myosin (kindly provided by Dan Kiehart) was diluted 1:250 in PBT1. After incubation with the appropriate primary antibody, the embryos were washed once with PBT1 and four times for 30 minutes each with BNT100 (PBS with 2% normal goat serum, 100 mM NaCl, 1% BSA, and 0.1% Tween20). Preabsorbed fluorescent secondary antibodies were diluted 1:250 in PBT0.1 (PBS with 0.1% BSA and 0.1% Tween-20) and embryos were incubated with them at room temperature for one to three hours. To visualize filamentous actin, embryos were stained for 20 minutes with either 0.165 µM bodipy-phalloidin or 0.165 µM rhodamine-phalloidin (Molecular Probes). After several washes in PBS-Triton, they were incubated for 3 minutes with 1 µg/ml Hoechst 33258 (Polysciences), a DNA-specific dye. The embryos were washed extensively in PBS-Triton and PBS before being mounted in Aquapolymount (Polysciences). Embryos were examined and photomicrographs made using a Bio-Rad MRC600 confocal microscope. Fig. 1. The actin-myosin network that forms in Drosophila embryos during cycle 14. In wild-type embryos (A), the array consists of approximately 6000 polygons of roughly equal size. Each polygon defines the area above a single somatic nucleus. Bar, 100 µm. At higher magnification, the wild-type array (B) shows a very regular configuration of polygons composed of uniformly thick actin interfaces. In embryos deficient for nullo (C) or sry-α (D), the array has interfaces of irregular thickness and the individual polygons are of variable size. Actin microfilaments were visualized by staining with FITC-labeled phallacidin. Bar in D (also applies to B and C), 10 µm. RESULTS nullo protein is blastoderm-specific In order to characterize the distribution of nullo protein, monoclonal antibodies to the protein were isolated. The antigen was a fusion protein consisting of glutathione S-transferase and the full-length nullo protein. Supernatants from two monoclonal lines recognized proteins from two- to three-hour-old embryos in the size range predicted for nullo (approximately 23 kDa). Both supernatants reacted with the same protein doublet of 25 and 26.5 kDa (Fig. 2A). The doublet was absent in protein preparations from embryos homozygous for a small deficiency of nullo (Fig. 2B and C), indicating that both proteins are encoded by the nullo gene and that both monoclonal lines are specific for nullo proteins. The difference in migration between the two forms of nullo is probably due to a post translational modification, since northern blots and sequence analysis predict only a single nullo product (Rose and Wieschaus, 1992). The temporal profile of nullo protein levels was determined by western blot analysis of single Ore-R embryos staged prior to homogenization (Fig. 1D). nullo protein was detectable between interphase of cycle 13 and the beginning of gastrulation. The amount of nullo protein is low in cycle 13 interphase embryos, increases greatly during the 13th division, and reaches an apparent peak in early cycle 14. Levels of nullo protein remain high during the initial slow phase of cellularization, when invagination of membrane is thought to depend on the incorporation of new membrane behind the furrow (Turner and Mahowald, 1976). Levels drop rapidly during the subsequent ‘fast’ phase and protein is barely detectable by the beginning of gastrulation. Both forms of nullo protein show similar kinetics of accumulation and disappearance. The pattern parallels that previously reported for the nullo RNA with a lag of about fifteen to twenty minutes. Intracellular localization of nullo proteins Antibodies to nullo were used for immunolocalization of the protein during syncytial and cellular blastoderm stages. To visualize the microfilament network independently, wild-type embryos were simultaneously stained with antibodies against myosin or with phalloidin. nullo protein is first detectable in whole-mount embryos during interphase of cycle 13 (Fig. 3A). At this stage, all detectable nullo protein is localized in the cortical cytoplasm of the embryo and is punctate or vesicular in nature. The protein is restricted to a region apical to the monolayer of nuclei, and appears to be associated with the plasma membrane. It does not co-localize with the actin caps that form above the interphase nuclei and instead appears to be restricted to the areas between the caps. When viewed from the surface, the resulting pattern of nullo distribution resembles a 1866 M. A. Postner and E. F. Wieschaus Fig. 2. Western blots of embryo extracts using nullo monoclonal antibody. (A) Extracts from 2- to 3-hour wild-type embryos. Lane 1 contains approximately 10 embryos; lane 2 contains approximately 5 embryos; and lane M contains unstained low molecular mass markers (Bio-Rad). The monoclonal antibody recognizes two proteins of approximately 25 and 26.5 kDa. (B) Each lane contains proteins extracted from a single cycle 14 embryo. The embryos were collected from a cross in which one-quarter of the embryos are deleted for the nullo locus. The embryos were randomly harvested during early cycle 14 and no attempt was made to pick normal or mutant embryos. The embryos in lanes 2, 4, 8 and 11 lack both nullo proteins and are assumed to be deleted for the nullo locus. (C) In this control experiment, each lane contains proteins extracted from a single wild-type embryo in cycle 14. As expected, two forms of the nullo protein were recovered from all embryos. (D) Wild-type embryos were carefully staged and harvested at precise stages, from the beginning of cycle 13 through early gastrulation. Each lane contains protein from a single embryo. Lanes 1 and 2, cycle 13, interphase; lanes 3 and 4, cycle 13, mitosis; lanes 5 and 6, cycle 14, precellularization; lanes 7 and 8, slow phase of cellularization; lane 9, beginning of fast phase of cellularization; lanes 10 and 11, end of fast phase; and lanes 12 and 13, gastrulation. The levels of the nullo protein are highest from the 13th division through the slow phase of cellularization. relatively regular network of interlocking rings that encompasses the entire embryo (Fig. 3A). The intensity of staining with the nullo antibody is weak at this stage. During the mitosis of cycle 13, nullo protein is localized within the pseudocleavage furrows that transiently invaginate between the dividing nuclei. Although actin underlies the entire plasma membrane during this stage, nullo appears to be associated only with invaginated membrane regions. When viewed from above (Fig. 3B), nullo forms elongated hexagons, each enclosing a mitotic nucleus. The intensity of nullo antibody staining at this stage is considerably greater than that in interphase of cycle 13. At the onset of cycle 14, when actin caps re-form and the hexagonal array of actin and myosin begins to resolve, nullo protein localizes to the bases of the caps (Fig. 4A,B,C,D). During the next ten minutes, as the network matures, nullo protein is colinear with the network and shows an intense and orderly punctate staining pattern (Fig. 4E,F,G,H). Closer analysis of the staining patterns within each hexagonal unit reveals that nullo and actin are distinguishable. When visualized with phalloidin or phallacidin, the hexagonal units are defined by uninterrupted ‘lines’ of filamentous actin. The ends of these lines meet to form the hexagon’s vertices (Rose and Wieschaus; see also Fig. 5). By comparison, nullo protein shows a discontinuous, punctate staining pattern. Many dots of nullo protein are aligned to give the hexagonal pattern. While there is nullo present along the sides of each hexagon, the highest concentration of nullo staining occurs at the vertices. In addition, some nullo protein is present in the apical regions above the nuclei, as well as in the cytoplasm just below the Fig. 3. Localization of the nullo protein in late syncytial embryos. When wild-type embryos at interphase of cycle 13 (A) are viewed from surface, the nullo protein forms a hexagonal network. The nullo protein localizes to the pseudocleavage furrows during the mitosis of cycle 13 (B). It forms a hexagonal array in cycle 14 (C). The absolute level of staining with the nullo antibody dramatically increases from interphase of cycle 13 to early cycle 14. To compare spatial patterns of distribution, all panels have been printed at the same intensity levels. Bar in C (also applies to A and B), 10 µm. nullo protein in Drosophila embryos 1867 Fig. 4. Cellularizing wild-type embryos double labelled with the nullo antibody (A,C,E,G,I,K,M,O,Q,S) and cytoplasmic myosin antibody (B,D,F,H,J,L,N,P,R,T). The first and second columns show surface views, the third and fourth columns show cross-sections from the same embryos. During conversion of the actin caps to a hexagonal network (A-D), the nullo protein is localized to the forming network (A,C). It maintains a localization to the leading edge of the invaginating membrane at the initiation of membrane invagination (E-H) and during slow phase (I-L). During the fast phase of cellularization (M-P), the levels of nullo protein present at the cellularization front decrease and the protein becomes dispersed throughout the cytoplasm. By the completion of cellularization (Q-T) nullo protein is almost undetectable. Bar in D (applies to entire figure), 10 µm. 1868 M. A. Postner and E. F. Wieschaus bases of the nuclei, where its staining pattern gives the impression of being vesicular. During the slow phase of cellularization (Fig. 4I,J,K,L), nullo protein continues to be primarily associated with the plasma membrane at and slightly apical to the invaginating furrow canal, where it maintains its initial distribution in a hexagonal array. In some confocal cross-sections, nullo staining appears as a line extending apically from the furrow canal. This line never extends more than half-way to the apical plasma membrane. Unlike actin, nullo is not localized to the non-invaginated plasma membrane on the apical surface, although some slight staining above background is observed in Fig. 5. Cellularizing wild-type embryos double labelled with the sry-α antibody (A,C,E,G,I,K,M,O) and phalloidin (B,D,F,H,J,L,N,P). The first and second columns show surface views; the third and fourth columns show cross-sections from the same embryos. During actin-myosin network resolution (A-D) and during slow phase of cellularization (E-H), sry-α protein localizes to the hexagonal array (see arrows in C and G). The levels of localized sry-α protein remain high at the end of slow phase (I-L) but decrease during the latter half of fast phase (M-P). The protein becomes more dispersed in the cytoplasm (M, O), although some staining remains in a hexagonal pattern (M). sry-a protein also localizes in a sphere above each nucleus (G and K, arrowheads) until the final stages of cellularization (G,K,) as well as maintaining a more general localization in the apical cytoplasm (G,K). Bar in D (applies to entire figure), 10 µm. nullo protein in Drosophila embryos 1869 the cytoplasm apical to the nuclei. All cytoplasmic staining observed at these stages is still punctate in appearance. As cellularization progresses into the fast phase (Fig. 4M,N,O,P), nullo protein maintains its localization to the cellularization front, but the intensity of staining is significantly diminished and more variable from embryo to embryo than in previous stages of cellularization. When viewed tangentially at the level of the furrow canals, the nullo protein continues to form a thin network of interconnecting hexagons. This is in contrast to the actin-myosin network, which thickens, resulting in a ring-like appearance of the individual units in the network. During the course of fast phase (Fig. 4Q,R,S,T), progressively Fig. 6. Localization of the nullo proteins in nullo and sry-α mutant embryos. Embryos deleted for nullo locus (E,F,G,H) and their heterozygous siblings (A,B,C,D) were double labelled using nullo antibody (A,C,E,G) and cytoplasmic myosin antisera (B,D,F,H). The specificity of the nullo antibody is demonstrated by its failure to stain nullo mutant embryos (E,G). In embryos lacking sry-α, (M,O), the nullo protein forms a network that is collinear with the actin-myosin hexagonal array: a relatively normal nullo network is seen everywhere except where the actinmyosin network is disrupted. Bar in D (applies to entire figure), 10 µm. 1870 M. A. Postner and E. F. Wieschaus less nullo protein is localized to the furrow canal and the majority of nullo protein becomes dispersed throughout the cytoplasm. The intensity of nullo staining continues to decline and is no longer detectable above background when cellularization is completed. Intracellular localization of the serendipity-alpha protein Since nullo and serendipity-alpha (sry-α) mutants have a common phenotype and both proteins have now been reported to localize to the hexagonal array of actin, a detailed compar- Fig. 7. Localization of the sry-α protein in nullo and sry-α mutant embryos. Comparison of embryos deleted for sry-α (E,F,G,H) and their heterozygous siblings (A,B,C,D) when double labelled with sry-α antibody (A,C,E,G) and phalloidin (B,D,F,H) confirm that the sry-α antibody is specific: sry-α heterozygotes have a hexagonal array of sry-α protein (A,C) whereas their mutant siblings show no such staining (E,G). Embryos deleted for nullo (M,N,O,P) and their heterozygous siblings (I,J,K,L) were also double-labelled with sry-α antibody (I,K,M,O) and phalloidin (J,L,N,P). The sry-α protein in nullo mutants (M,O) does not form a hexagonal network at the level of the cellularization front. Bar in D (applies to entire figure), 10 µm. nullo protein in Drosophila embryos 1871 ison of the intracellular distributions of these two proteins is merited. Mice were immunized with a truncated version of the sry-α protein and supernatants from several of the resulting monoclonal lines reacted with a protein from two- to threehour fly embryos of approximately 58 kDa, the size of sry-α as defined by polyclonal antisera (Schweisguth et al., 1990). The specificity of the monoclonal antibodies was shown by the failure of embryos deleted for the sry-α gene to exhibit any of the staining patterns described (see Fig. 7E,G). Although the sry-α protein has been reported to be present in cycle 13 embryos (Schweisguth et al., 1990), our antibodies only detect sry-α protein above background staining during the formation of the hexagonal network in early cycle 14. As soon as the hexagonal network of actin is present, sry-α forms a colinear array (Fig. 5A,B). Like actin, the sry-α array is composed of ‘lines’ of sry-α staining with each line forming the side of a hexagon. However, the lines of sry-α protein often do not meet at the hexagon vertices and sry-α is relatively depleted there. This staining pattern is in marked contrast to the dots of nullo staining that compose the sides and, most prominently, the vertices of the hexagons. During early cellularization stages (Fig. 5C,D), sry-α appears to be associated with the entire plasma membrane. The sry-α protein shows an additional distinctive localization that is not shared by actin, myosin or nullo: the protein is localized above each cycle 14 nucleus in a small spherical structure of unknown origin or significance (Fig. 5G,K). This sry-α staining may be associated with the centrosomes or the microtubule arrays located apical to the nuclei at these stages (Whitfield et al., 1988; Kellogg et al., 1989). Several basic features of sry-α localization are maintained throughout the slow phase (Fig. 5I,J,K,L,M,N,O,P) and much of the fast phase (Fig. 5Q,R,S,T) of cellularization. The sry-α protein remains associated with the invaginating actin-myosin hexagonal array until late in the fast phase. Although the actinmyosin network assumes a ring-like appearance during fast phase, sry-α protein continues to form interlocking hexagons that are composed of lines of sry-α staining (compare Fig. 5M and 5N). In this regard, sry-α resembles nullo, since neither protein co-localizes with the rings. It should be noted, however, that sry-α protein remains localized to the furrow canal during more advanced stages of cellularization than nullo does. During cellularization, the association of sry-α with the plasma membrane is limited to the furrow canal and the lateral membranes just apical to it. In addition, sry-α protein continues to be localized to a spherical structure above each nucleus. No sry-α protein seems to be specifically localized to the apical plasma membrane, but the protein is present in the cytoplasm above and, to an increasing extent, below the nuclei. Towards the end of the fast phase of cellularization (Fig. 5S,T), the localizations of sry-α protein to the cellularization front and to the spherical structures above the nuclei are lost and most of the sry-α protein becomes dispersed throughout the cytoplasm. The intensity of sry-α antibody staining diminishes rapidly during the final stages of cellularization, although sry-α protein remains detectable in gastrulating embryos (data not shown) until the onset of germ band extension. Localizations of nullo and sry-α in mutant embryos The similarities in their mutant phenotypes suggest that nullo and sry-α proteins may be components of the same develop- mental pathway. Previous experiments have shown that the genes are transcriptionally independent (Rose and Wieschaus, 1992). To determine if either protein regulates the translation or intracellular localization of the other, embryos derived from either nullo or sry-α stocks were stained with antibodies to the reciprocal protein. By approximately 10 minutes into cycle 14, both nullo and sry-α mutant embryos can be distinguished from sibling embryos: they exhibit an abnormal array of actin and myosin that has unusually thin and thick portions and some interfaces that are completely disrupted. All cycle 14 embryos lacking sry-α clearly contain nullo protein. The protein forms a roughly hexagonal network in sry-α mutant embryos with some disrupted interfaces (Fig. 6M,N,O,P). The discontinuities in nullo staining completely match the disruptions seen in the actin-myosin network. Therefore, nullo protein continues to be coincident with actin and myosin in the sry-α mutant background. Some sry-α embryos appear to have fewer ‘dots’ of nullo staining than wild-type embryos. It is not clear whether this decreased intensity of staining is due to an overall decrease in levels of nullo protein or a slightly compromised ability of the protein to be properly localized. Overall, however, the distribution of nullo protein is remarkably normal in sry-α mutants. In contrast, absence of nullo activity has a more striking effect on the localization of sry-α protein. While phenotypically nullo embryos contain large pools of sry-α protein, its distribution pattern is altered. sry-α protein fails to co-localize with the leading edge of the cellularization front and and the well defined lines of sry-α staining normally associated with the actin-myosin array are absent (Fig. 7M,O). Instead, the entire cytoplasmic region between the nuclei shows a low level of sry-α staining. Localization of sry-α protein to the spherical structure above each nucleus persists, however, suggesting that nullo activity is specifically required for association of sry-α protein with the actin-myosin network. DISCUSSION Translation and post-translational modification of the nullo protein Transcription of nullo RNA was previously demonstrated to be very tightly regulated (Rose and Wieschaus, 1992). The nullo RNA is expressed only for a brief period during the Drosophila life cycle: developmental Northern blots revealed the presence of the nullo transcript in RNA samples from 0- to 4-hour embryos but not in any other developmental stages. In RNA in situs to whole mount embryos, the transcript was detectable from the beginning of cycle 11 through the slow phase of cellularization. Within this short interval, large amounts of nullo transcript rapidly accumulate, reaching a maximum level during the division between cycle 13 and 14. As soon as cycle 14 begins, levels of nullo RNA plummet. While accumulation of the nullo transcript occurs uniformly throughout the embryo, nullo degradation does not; a reproducibly banded pattern of the nullo transcript is visible in early cycle 14. Both developmental western blots of individual, precisely staged embryos and antibody staining of whole-mount embryos indicate that the dynamics of nullo protein expression 1872 M. A. Postner and E. F. Wieschaus mirror those of its RNA. By western analysis, the nullo protein is detectable from the start of cycle 13 through the beginning of gastrulation. The highest levels of nullo protein are detected in embryos in early cycle 14, just prior to and during the slow phase of cellularization. The levels of nullo protein then drop rapidly during the fast phase of cellularization. The protein expression pattern thus closely resembles the transcription pattern with a translational lag of no greater than twenty minutes. This comparison implies that nullo protein has a consistently short half-life: nullo protein is constantly being degraded and replaced with newly synthesized protein until lack of transcript prevents its replacement. An additional mechanism of specific degradation late in cellularization cannot be ruled out. Independent monoclonal antibodies specifically recognize two forms of the nullo protein in crude protein preparations from early embryos. Preliminary results suggest that the size difference between the two forms reflects a differential phosphorylation, since treatment with bacterial alkaline phosphatase causes the conversion of the slower migrating form into the faster migrating form (Postner, 1993; and unpublished observations). The significance of this modification is unclear. We did not detect pronounced differences in the ratio of phosphorylated to unphosphorylated nullo protein during the entire window of nullo protein expression. The nullo and sry-α proteins are components of the hexagonal network During early cycle 14, nullo and sry-α proteins both colocalize to the hexagonal array of actin and myosin. They maintain this association at least until the fast phase of cellularization (when the cleavage furrow has invaginated about half its final depth). A more thorough biochemical analysis is required to determine whether either protein interacts directly with the actin cytoskeketon. The distributions of the two proteins within the network are not identical. The sry-α protein is found in lines running along the sides of the hexagons. These lines rarely reach the hexagonal vertices and little sry-α protein is detectable within the vertices. This contrasts with the distribution of nullo protein, which shows a punctate pattern throughout the hexagonal array. nullo appears at regular intervals along the sides of the hexagons and is particularly abundant at the hexagonal vertices. The punctate staining pattern is observed from the earliest stages when the protein can be detected and suggests a vesicular localization at least during synthesis or transport to the surface. The potential myristoylation codon at the amino terminus of the nullo protein (Rose and Wieschaus, 1992) might provide a mechanism for associating the protein with transport vesicles and ultimately with the plasma membrane. The discovery that the Drosophila virilis homologue of the nullo protein contains an equally favorable potential myristoylation codon amidst an otherwise divergent amino terminus (E. Schejter, personal communication) strongly suggests that this sequence is important functionally. If the nullo protein is myristoylated in vivo, nullo might also provide a crucial link between the actin hexagonal network and the plasma membrane. During interphase of cycle 13, nullo forms a network of interconnecting rings of protein that is associated with the surface regions between the actin caps. This hexagonal pattern precedes formation of the actin hexagonal array and might therefore provide a spatial cue, localizing other proteins into a hexagonal array following the 13th mitosis. Since an actinmyosin network, albeit abnormal, still forms in nullo mutants, nullo protein cannot provide the only bias or scaffold for recruitment of microfilaments and myosin into the hexagonal pattern. However, other components of the array might be completely reliant on cycle 13 nullo localization for their proper distributions; lack of such proteins might cause the hexagonal array that forms in cycle 14 to be unstable and result in the disruptions seen in embryos lacking the nullo gene. Given the similarities between the nullo and sry-α phenotypes, an obvious possibility is the sry-α protein. In cellularizing embryos mutant for the nullo locus, the sryα protein is localized to a sphere above each nucleus and distributed throughout the apical cytoplasm just as in wild-type embryos. However, the protein specifically fails to form a discrete hexagonal network. Instead, sry-α protein shows a cytoplasmic localization that is indistinguishable from that seen in more apical regions. The failure of sry-α protein to form even a disorganized hexagonal array in embryos lacking the nullo gene indicates that sry-α is dependent upon nullo for its localization to the hexagonal network. On the other hand, nullo protein still co-localizes with the actin network in sry-α mutants, even though that network is disrupted. This suggests that the relationship between the loci is not reciprocal and that sry-α protein is functionally downstream of nullo. The relatively minor effect of sry-α protein on final nullo distribution is probably indirect: a result of the instability of the actinmyosin network in sry-α mutants. In this model the nullo phenotype would result in part from the failure of sry-α to localize to the network and maintain its stability. This proposal does not account for the difference in severity between the two null phenotypes. Since more disruptions of the hexagonal network occur in nullo mutant embryos than in sry-α mutant embryos, the nullo phenotype cannot be due in its entirety to a mislocalization of sry-α protein. In addition to facilitating the proper localization of the sry-α protein, the nullo protein must further contribute to the stability of the hexagonal array. It could do so indirectly by recruiting other stabilizing molecules to the network or directly by binding, and thus anchoring, multiple components of the network. We thank Dan Kiehart for the gifts of antibodies and Marty Marlow of the Princeton’s monoclonal facility for establishing the antibody lines. Joe Goodhouse, Mark Peifer and Romy Knittel provided valuable technical advice on confocal microscopy, fusion proteins and monoclonal antibody screening, respectively. 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