replication with aphidicolin failed to prevent ectopic expression of the homeotic gene Ultrabithorax in Pc mutants. Thus, even in the absence of DNA replication,.
Development 110, 1319-1325 (1990) Printed in Great Britain © The Company of Biologists Limited 1990
1319
Blocking cell division does not remove the requirement for Polycomb function in Drosophila embryogenesis
ALEX P. GOULD, ROBERT Y. K. LAI, MICHAEL J. GREEN and ROBERT A. H. WHITE Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3 DY, UK
Summary The Polycomb (Pc) gene is required from the extended germ band stage onwards, to maintain spatially restricted patterns of homeotic gene expression. It has been thought to be involved in the 'stable inheritance of the determined state'. In this paper, we have tested the notion that the Pc gene is required specifically during or after DNA replication to enable the stable transmission of states of gene activity. We found that arresting cell division using the string mutation or blocking DNA replication with aphidicolin failed to prevent ectopic
expression of the homeotic gene Ultrabithorax in Pc mutants. Thus, even in the absence of DNA replication, Pc is required to maintain spatially restricted patterns of homeotic gene expression. The role of the Pc gene product in the stable repression of homeotic gene transcription is discussed.
Introduction
Frederick, 1983; Struhl and Akam, 1985; Casanova and White, 1987). The implication is that the 'Pc group' genes provide a special mechanism for the inheritance of states of gene activity from mother to daughter cells. More specifically, it might be expected that this function would be required during or after DNA replication. A close link between cell division and the ectopic expression of homeotic genes is supported by some circumstantial evidence. For instance, the initial ectopic expression of the homeotic gene Ultrabithorax (Ubx) in Pc mutants appears as isolated doublets of cells suggesting a prior requirement for cell division and also the ectopic expression is most pronounced and uniform in the ventral nervous system which is the most mitotically active tissue in embryonic development (Casanova and White, 1987; Campos-Ortega and Hartenstein, 1985). Furthermore, in the case of esc mutations, the ectopic expression of Ubx transcripts occurs at about 6h of development, which would be consistent with a derepression occurring at the S phase of cell cycle 15 (Struhl and Akam, 1985). This S phase would constitute the first DNA replication after the establishment of the Ubx expression pattern in cycle 14 (Akam and Martinez-Arias, 1985). In this paper, we test the idea that Pc is required to maintain the spatial restriction of homeotic gene expression during or after DNA replication. We examine the effect of preventing DNA replication in cycle 15, either by directly blocking replication using the inhibitor aphidicolin (Ikegami et al. 1978) or by using the string (stg) mutation to arrest the cells of the developing embryo at the G2/M transition in cycle 14
In Polycomb (Pc) homozygous mutant embryos, homeotic gene expression is initially normal but during the extended germ band stage the spatial restriction breaks down resulting in widespread ectopic expression of homeotic gene products (Beachy et al. 1985; White and Wilcox, 1985; Wedeen et al. 1986; Casanova and White, 1987). The Pc gene, acting together with other genes of the 'Pc group' e.g. esc, Pel, sxc, (Jiirgens, 1985; Lewis, 1978; Struhl, 1981; Duncan, 1982; Ingham, 1984), appears to constitute a maintenance machinery ensuring the preservation of the initial domains of homeotic gene activation (Struhl, 1981; Denell and Frederick, 1983; Struhl and Akam, 1985). These domains are established primarily by the regulation of the homeotic genes by members of the gap class of segmentation genes (White and Lehmann, 1986; Harding and Levine, 1988; Irish etal. 1989) However, the gap genes are only transiently expressed in the simple block patterns that define regions within the embryo (Gaul et al. 1987; Tautz et al. 1987). Thus the 'Pc group' genes appear to be required to ensure that this transient expression has a lasting influence. There are many systems where states of gene activity are maintained after the disappearance of the signals responsible for their initiation (Weintraub, 1985) and the 'Pc group' genes offer the possibility of investigating the mechanisms involved. Several authors have attributed the defect in Pc mutants to an 'instability in the clonal transmission of the determined state' (Struhl, 1981; Denell and
Key words: Polycomb, Ultrabithorax, homeotic genes, gene regulation.
1320 A. P. Gould and others (Edgar and O'Farrell, 1989; Jiirgens et al. 1984). If the Pc machinery is required for the stable transmission of gene activity status through DNA replication, then preventing the S phase of cycle 15 should remove the requirement for Pc function and hence there should be no ectopic homeotic gene expression. However, we observe, in both circumstances of cell cycle block, no effect on the ectopic expression of the homeotic gene Ubx in Pc mutants.
Materials and methods
Fly stocks The wild-type stock used was Oregon R. The homozygous Pc mutant embryos were collected from a balanced Pc3 heterozygous stock (Duncan and Lewis, 1982). For the stg mutant embryos, we used stg7B69, one of the strongest stg alleles available (Edgar and O'Farrell, 1989). Pc stg chromosomes were constructed by recombination betweeen Pc3 and stg7B69 chromosomes. Embryos were collected from balanced heterozygous stocks and the homozygous stg embryos were clearly recognisable after cycle 14 by the small number and large size of their cells and nuclei. The esc6 homozygous embryos derived from esc6 homozygous mothers were g e n e r a t e d by crossing esc6/C\>.;
P[ry+;
esc+
F61],ry506/ry506
females with escS06/Cy O; ry /ry males and then crossing inter se the homozygous esc6; ry progeny (Frei et al. 1985). The esc6 mutation is an apparent null (Struhl, 1981).
Inhibition of replication by aphidicolin Embryos were collected and aged at 25 °C, dechorionated in bleach and thoroughly rinsed in water. They were permeabilized by swirling gently for 2min on the interface of a 1:1 mixture of Schneider's Drosophila medium (Gibco-BRL) and octane (Aldrich). The embryos were then transferred onto the surface of 2 ml Schneider's medium±aphidicolin in small vials and the octane was blown off gently with a stream of moist air. Aphidicolin was used at lO/igml"1, diluted from a stock of lmgml""1 in dimethyl sulphoxide.The vials were capped to prevent the embryos drying and were incubated at 25 °C. At the end of the incubation the embryos were lifted off the surface of the medium with a spatula and fixed and devitellinized for immunolabelling. For inhibition of replication in wild-type embryos, the collection was for 1 h followed by ageing for 2h and incubation for 7h. For the Pc embryos, the collection was for 2 h followed by ageing for 2 h and incubation for 5h.
Results
The onset of ectopic expression We detect the first ectopic expression of Ubx protein in Pc mutants at the extended germ band stage (Fig. 1). More precisely, it appears early in stage 11 (CamposOrtega and Hartenstein, 1985) during the period when the epidermal cells are undergoing the mitosis of cycle 15 (Fig. 1C). As transcription of the 75 kb Ubx gene is likely to take about an hour (Hogness et al. 1985), this timing of onset of protein expression would be consistent with derepression of the Ubx gene during the S-phase of cycle 15. Note in Fig. ID that the ectopic expression often appears in cell doublets, suggesting a correlation between the appearance of Ubx protein expression and cell division. The gross pattern of the onset of ectopic Ubx expression is fairly reproducible from embryo to embryo although, at the single cell level, the pattern is variable and is not symmetrical about the midline (Fig. IE). Aphidicolin can block embryos in the S phase of cycle 15 The Drosophila embryo undergoes 13 cell cycles as a syncytium with the nuclear divisions being synchronous or nearly so. Cellularization occurs during cycle 14 and most epidermal cells undergo a further two divisions during the remainder of embryogenesis (CamposOrtega and Hartenstein, 1985). These two divisions, mitosis (M)14 and M15, are asynchronous and for M14 it has been shown that cells divide in groups (mitotic domains) according to a rigid and highly patterned regime (Foe, 1989). Cells thus vary in the length of their cycle 14 and this variation is thought to occur in the G2 phase as neither cycle 14 nor 15 possess an appreciable Gi (Edgar and O'Farrell, 1989). Aphidicolin is a highly specific inhibitor of DNA replication (Ikegami et al. 1978). If injected prior to cellularization, aphidicolin blocks DNA replication but also arrests development due to failure to cellularize the blastoderm (Raff and Glover, 1988). To block thefirstS phase to occur after cellularization (S15) embryos were permeabilized with octane and then incubated in Schneider's medium containing lO^gml" 1 aphidicolin. Fig. 2A shows an embryo arrested in S15; it has the cell density characteristic of early stage 11 (i.e. prior to M15, see Fig. 1A) but by its age and morphology it should already have completed cycle 15.
Immunolabelling The monoclonal antibody FP3.38 was used to detect the Ubx protein products (White and Wilcox, 1984). The whole-mount embryo labelling protocol was adapted from Mitchison and Sedat, 1983 (Mitchison and Sedat, 1983) with a 40min fixation and devitellinization at room temperature. Rabbit anti-mouse immunoglobulins conjugated to horseradish peroxidase (Dako Ltd) were used as a second antibody. The enzyme was visualized using a developing solution containing PBS, 0.1 % Tween 20, 250 ^g ml" 1 diaminobenzidine hydrochloride, 0.6% nickel ammonium sulphate and 0.06% hydrogen peroxide. The photographs were taken on a Zeiss Axiophot microscope using Ilford Pan F film.
Blocking replication in Pc mutants Embryos derived from a Pc/+ stock were treated with aphidicolin to block replication in S15. Embryos were obtained that were arrested in cycle 15 but which exhibited clear ectopic expression of Ubx protein. Fig. 2B shows an embryo with ectopic Ubx expression but with a nuclear density indicative of a block prior to M15. Thus in the absence of DNA replication, the Pc function is still required to prevent ectopic expression of Ubx.
Polycomb function
md mx
1321
Ib
B
mx md
Ib
md mx
md mx
Ib
Fig. 1. The timing and pattern of ectopic Ubx protein expression in Pc mutants. A and B are wild-type embryos. A is an early stage 11; B is late stage 11 after the epidermal cells have undergone mitosis 15. Note the increase in the number of nuclei per metamere. C to E are homozygous Pc embryos. C illustrates the onset of ectopic Ubx expression. The epidermal cells are undergoing mitosis 15 (cf. A and B). Isolated ectopic expression extends forward to the gnathal segments. D is a higher magnification of a later embryo, after germ band retraction. The isolated doublets of ectopic Ubx expression in the gnathal segments are indicated (arrows). E is a ventral view of a late stage 11 embryo showing the pattern of ectopic Ubx expression. The numbers refer to parasegments. mb, mandibular segment; mx, maxillary segment; Ib, labial segment.
Pc stg mutants Although the aphidicolin experiment apparently gave a clear result, it is difficult to be certain that every cell in a developing embryo is successfully blocked in S15. Therefore, to confirm this result, we used the stg mutation to completely block cell division in cycle 14. Homozygous stg 9 embryos are arrested at the G 2 /M transition in cycle 14 and exhibit a total block of DNA replication in all cells except the polyploid cells of the amnio-serosa (Edgar and O'Farrell, 1989). Nevertheless, these mutant embryos continue their development through to late embryogenesis and are able to secrete cuticle. Fig. 3A shows the Ubx protein distribution in a stg homozygous mutant embryo and it is noteworthy that the Ubx expression in these arrested embryos is rather normal, both in the general pattern and in its
temporal development. The Ubx proteins are expressed in their characteristic domain from parasegments 5-13 with normal modulations in level between and within parasegments. The latter is interesting as individual parasegments contain several mitotic domains, at least for M14 (Foe, 1989), and it could have been imagined that the patterning of Ubx expression across the parasegment is dependent on the heterogeneous patterns of cell division. However, in these stg embryos we see a relatively normal intra-parasegmental patterning despite the absence of M14 and M15. Furthermore, in wild-type embryos, Ubx expression is strong in the epidermis and mesoderm during the extended germ band stage but then after germ band retraction it becomes largely restricted to the ventral nervous system. A similar development of the expression
1322 A. P. Gould and others on the one hand, removes the circumstantial argument for the doublets implying a causal link between cell division and ectopic expression. On the other hand, it indicates a similarity of status between neighbouring pairs of cells that we do not understand.
mx
mx
5
6
B Fig. 2. Distribution of Ubx protein in wild-type and Pc embryos after blocking of replication in cycle 15 by aphidicolin. (A) Wild-type aphidicolin-treated embryo. Despite the well developed gnathal buds (mx indicates the maxillary bud) and the retracting germ band indicative of stage 12, the number of nuclei per metamere corresponds to early stage 11 prior to mitosis 15 (see Fig. 1 A and B) (B) Aphidicolin-treated Pc homozygous embryo. Ectopic Ubx expression is observed in an embryo whose nuclear number indicates a block in cell division prior to mitosis 15. pattern appears to take place in stg mutant embryos (Fig. 3C) indicating that the state of activity of the Ubx gene can alter in the absence of DNA replication. Using Pc stg double mutants we can test whether blocking cell cycling at the G 2 /M boundary in cycle 14 removes the requirement for Pc function. The result is that it does not and Fig. 3B and C show Pc stg homozygous embryos with clear ectopic expression of Ubx protein. Other homeotic genes behave similarly and Pc stg mutant embryos show ectopic expression of Antennapedia and Sex combs reduced products (data not shown). Doublets One of the reasons (see Introduction) for thinking that cell division and Pc function might be closely linked is the appearance of the ectopic Ubx expression in Pc mutants in isolated doublets of cells (Fig. ID) (Casanova and White, 1987). What is the significance of these doublets if, as we have shown above, Pc function is still required in the absence of cell division? Fig. 3C illustrates an interesting twist to this argument as it appears that the ectopic Ubx expression still occurs in doublets in Pc stg double mutants where postblastoderm cell divisions are completely blocked. This result,
Ubx expression in esc mutant embryos In order to compare the onset of Ubx expression in Pc mutants with that in mutants of other genes in the 'Pc group', we examined Ubx expression in homozygous esc mutant embryos derived from homozygous mutant mothers (Struhl, 1981). The occurrence of ectopic Ubx expression in such embryos has been reported previously (Struhl and Akam, 1985) and here we would simply like to contrast the uniformity of the ectopic expression in these esc mutant embryos (Fig. 3D) with the heterogeneous expression seen in Pc mutant embryos. Ectopic expression extends both anterior and posterior to the normal domain of expression. In the region of parasegments 2-5, the expression exhibits a simple repeat pattern similar to that normally seen in parasegments 7-12. Thus, the normal repression of Ubx expression in posterior compartments by engrailed (Martinez-Arias and White, 1987) may also operate in the parasegment 2-5 region in esc mutant embryos. It appears that the variability and heterogeneity of the ectopic Ubx expression in Pc embryos is not a general characteristic of homeotic gene expression in all 'Pc group' mutants. At present we do not know whether this indicates a clear functional difference between esc and Pc or rather that the pattern in Pc homozygotes, derived from heterozygous mothers, is complicated by the presence of the maternal Pc contribution (Denell, 1978). Discussion
The expression patterns of homeotic genes are established at the cellular blastoderm stage by a combination of controls from the segmentation genes (see Akam, 1987 for review). Although homeotic gene expression is dynamic, and is subject to a variety of modifications at different times in development and in different tissues, the initial expression domains are roughly preserved throughout development. The genes that are responsible for the initial specification of much of the pattern, the gap and pair-rule loci, are, however, only transiently expressed. The 'Pc group' genes seem to be required for this transient expression to leave a permanent imprint. Here, we have shown that blocking DNA replication immediately after the establishment of the Ubx expression pattern does not prevent ectopic expression of Ubx protein in Pc mutants. The same result was obtained whether DNA replication in cycle 15 is prevented by specific blocking with aphidicolin or if the stg mutation is used to arrest cell cycling in the embryo at the G2/M transition of cycle 14. Thus the function of the Pc gene is not merely to act as a component of a machine that ensures the stable transmission of states of
Polycomb function
1323
\
' 5 » 6
B
5
6
3 I 4 I 5 I
6
Fig. 3. Ubx protein expression in stg, Pc stg, and esc embryos. (A) Homozygous stg mutant embryo. Note large nuclei and the small number per metamere. The Ubx expression pattern looks rather normal. (B) Homozygous Pc stg mutant embryo. The number and size of nuclei clearly identifies this genotype. Ectopic Ubx expression extends forward from parasegment 5. The focal plane concentrates on the ectopic expression leaving the lateral epidermal nuclei largely out of focus. (C) Germ band retracted Pc stg homozygous mutant embryo. Strong Ubx expression is largely restricted to ventral cells. The arrows indicate isolated doublets of ectopic Ubx expression. (D) Homozygous esc mutant embryo derived from a homozygous esc mutant mother. Note the rather uniform pattern of ectopic expression extending anteriorly from parasegment 5. The position of parasegment borders is indicated by reference to the parasegmental grooves.
gene activity through the process of DNA replication. This argues against the 'inheritance machine' model for the function of the lPc group' genes as a whole and instead points to the involvement of Pc as a specific regulator of homeotic gene expression. In particular, our results emphasize the role of Pc in the maintenance of transcriptional repression. What role does transcriptional repression play in the control of homeotic gene expresson during early development? Lewis originally postulated an important role for Pc as a repressor molecule and suggested that the initial position-specific expression of homeotic genes was orchestrated by a gradient of the Pc protein (Lewis, 1978). Since the relegation of the 'Pc group' genes to a maintenance role (Struhl, 1981; Struhl and Akam, 1985), repression has been accorded less prominence. A recent model for the regulation of the bithorax complex suggests that the complex is initially packaged into inactive chromatin domains and that expression is dependant upon the activation of particular domains (Peifer et al. 1987). These domains are activated in a position-dependant manner and as one proceeds from the anterior to the posterior of the embryo more domains are opened thus allowing transcriptional regulators access to the parasegmentspecific enhancers located within each domain. In this 'open for business' model, it is the position-specific activation of chromatin domains that provides the basis for the patterned expression of homeotic genes. Where
does the 'Pc group' repression function fit into this scheme? One might have suggested that this function is required to maintain the closed state of the inactive domains through cell division. However, the results presented here argue against a specific role for the Pc group genes in inheritance. Indeed, in a model where the expression domains of homeotic genes are controlled by activation from a repressed 'ground state', there appears to be little obvious need for the 'Pc group' repression function. In contrast to this 'open for business' model, we would like to propose an alternative that emphasizes the role of repressive transcriptional control. During cycle 14, regulatory elements in the homeotic gene complexes would be accessible for binding by transcription factors encoded by the segmentation genes. These regulatory interactions apparently can be positive and negative. In the case of the Ubx gene, we have argued that the gap gene hunchback plays a critical role in defining the domain within which other regulators may operate to influence levels of expression (White and Lehmann, 1986). Recently there has been further evidence for the overriding repressive effect of hunchback as persistence of the maternal hunchback expression in oskar mutants represses Ubx expression in a region where Ubx is normally active (Irish et al. 1989). However, the expression of hunchback is transient (Tautz et al. 1987) and so there is a .need for a mechanism to maintain this repressive state after the
1324 A. P. Gould and others disappearance of the hunchback product. We suggest that homeotic genes that have been repressed by gap gene products are fixed in an inactive state by the 'Pc group' genes. An important role for specific repression, in generating the initial expression patterns of gene expression in embryogenesis, has also been suggested by experiments where blocking protein synthesis elicits widespread ectopic transcription of the segmentation gene fushi tarazu (Edgar et al. 1986). Are homeotic genes inaccessible to transcription factors outside their specific activity domains? The Antennapedia gene can be activated outside its normal domain by the heat-shock-induced expression of a chimaeric Ubx-Deformed homeoprotein (Kuziora and McGinnis, 1989). However, it is interesting that the sensitive period for ectopic activation of Antennapedia is confined to the cellular blastoderm or early gastrulation stage. This suggests that the Antennapedia gene is initially accessible to regulation outside its normal domain and furthermore that this accessibility ceases around the time of the onset of 'Pc group' gene function. This would seem at odds with the 'open for business' model and rather suggests that the regulatory elements of homeotic genes are initially open and not closed. How do the 'Pc group' genes act to maintain repression? The Pc gene product can associate with chromatin and has been shown to bind to about 60 sites on salivary gland polytene chromosomes (Zink and Paro, 1989) although it is not thought capable of binding DNA directly (R. Paro, personal communication). It has been proposed that the 'Pc group' genes may inactivate homeotic genes by packaging them as heterochromatin (Reuter et al. 1990; Gaunt and Singh, 1990). The predicted sequence of the Pc protein would support this idea as it shows homology to a suppressor of variegation gene product likely to be involved in heterochromatin formation (R. Paro, personal communication). The formation of heterochromatin is not the only conceivable way to shut down homeotic genes. The regulation of genes of the bithorax complex clearly involves interactions between several distant regulatory regions (Bender et al. 1983; Beachy et al. 1985; White and Wilcox, 1985; Karch et al. 1985). The Pc group genes might be involved not so much in shutting down whole domains but may act more locally to stabilize a repressive configuration of regulatory elements. The Pc gene is not, however, an obligatory participant in all forms of homeotic gene repression and not all repressive interactions are alleviated in Pc or 'Pc group' mutants. For instance, ectopic expression does not appear as a uniform blanket but rather the heterogeneous pattern indicates continued negative regulation by segment polarity and dorsoventral genes. For example in Fig. 3D the intrametameric modulation suggests the persistence of the repression by engrailed normally associated with the Ubx expression pattern in parasegments 7-12 (Martinez-Arias and White, 1987). Furthermore, the down regulation of Ubx expression by other
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