Overexpression of oskar directs ectopic activation of nanos and presumptive pole cell formation in Drosophila embryos

Cell, Vol. 70, 849-859,

September

4, 1992, Copyright

0 1992 by Cell Press

Overexpression of oskar Directs Ectopic Activation of nanos and Presumptive Pole Cell Formation in Drosophila Embryos Jeffrey L. Smith, Joan E. Wilson, and Paul M. Macdonald Department of Biological Sciences Stanford University Stanford, California 94305

Summary In Drosophila, a small group of maternal effect genes, including oskar, defines a shared pathway leading to the provision of two determinants at the posterior pole of the embryo. One determinant is the posterior body patterning morphogen nanos, and the other directs germ cell formation. Overexpression of oskar causes the shared pathway to be hyperactivated, with excess nanos activity present throughout the embryo and a superabundance of posterior pole cells. In addition, presumptive pole cells appear at a novel anterior position. Strikingly, formation of these ectopic pole cells is enhanced in nanos mutants. This observation may reflect competition between nanos and the germ cell determinant for a shared and limiting precursor. Introduction A common strategy for creating polarity or asymmetry within a single cell is to distribute components of the cytoplasm unevenly prior to division (Davidson, 1986; Horvitz and Sternberg, 1991). Such partitioning can be directed, with identifiable molecules or structures localized to defined positions. In some cases, the localized molecules have been shown to be directly involved in specification of pattern or cell fate. In other cases the presence of localized molecules suggests that as yet unidentified determinants could be similarly positioned. Prominent examples of the former type include two morphogens involved in specification of anteroposterior body pattern in Drosophila. The products of the bicoid (bed) and nanos (nos) genes are largely responsible for anterior and posterior body patterning, respectively, and each is prelocalized as mRNA to the appropriate pole of the egg during oogenesis (Frigerio et al., 1986; Frohnhdfer and Nijsslein-Volhard, 1987; Berleth et al., 1988; Lehmann and Niisslein-Volhard, 1991; Wang and Lehmann, 1991). Another example, encompassing a wide phylogenetic range, is the specification of germ cells from a localized region of the egg. Although the actual germline determinants are not known, their presence is marked by the appearance of cytoplasmic inclusions, called P granules in Caenorhabditis elegans (Strome and Wood, 1982), polar granules in Drosophila (Mahowald, 1962) and nuage in a variety of species (Kerr and Dixon, 1974; Eddy, 1975; Mahowald, 1977). Among the examples of molecules specifically partitioned within the egg cytoplasm, the Drosophila posterior body patterning morphogen and germ cell determinant(s) are interesting and unusual in that a common mechanism

directs their localization to the posterior pole. This shared mechanism is defined by a set of eight “posterior group” maternal effect genes: cappuccino (cap@, spire (spir), staufen (stau), oskar (osk), vasa (vas), t&or (tud), valois (v/s), and mago nashi (Schiipbach and Wieschaus, 1986; Boswell and Mahowald, 1985; Lehmann and NiissleinVolhard, 1986; Manseau and Schiipbach, 1989; Boswell et al., 1991). Mothers homozygous mutant for any one of these genes produce embryos that are defective in abdominal body patterning and fail to form pole cells(the germline precursors). These two defects represent two branches of the posterior pathway. The branch leading to posterior body patterning further requires the genes nos (the determinant) andpumilio @urn), a gene suggested to reposition nos (Lehmann and Ntisslein-Volhard, 1988) or to stabilize or activate nos (Macdonald, 1992). It is not known if the other branch, leading to germ cell formation, requires additional genes. However, neither nos norpum appears to be necessary, since pole cells form in both mutants(Lehmann and Niisslein-Volhard, 1991, 1988). Progression along this shared pathway is reflected in the accumulation of the RNA and/or protein products of several posterior group genes, stau, osk, and vas, at the posterior pole of the developing oocyte. By examining how different posterior group genes disrupt the posterior localization of these RNAs and proteins, the positions of most genes in the pathway have been defined (Hay et al., 1988, 1990; Lasko and Ashburner, 1988, 1990; Manseau and Schiipbach, 1989; Kim-Ha et al., 1991; Ephrussi et al., 1991; St Johnston et al., 1991; Golumbeski et al., 1991). Posterior accumulation appears to occur in a stepwise fashion; each localized component is required for the localization of subsequent components. Once localized, the components remain at the posterior pole into embryogenesis. Continued posterior localization of these components is paralleled by a persistent requirement for the genes that encode them. Temperature shift experiments using conditional alleles reveal requirements for osk and vas extending from the time their products become localized until egg deposition (Lehmann and Ntisslein-Volhard, 1986, 1991). Similar experiments with stau establish a requirement extending from about the time of posterior stau protein localization intoembryogenesis(St Johnston et al., 1991). These observations reinforce the notion, originally suggested by the absence of polar granules in many posterior group mutants (Boswell and Mahowald, 1985; Lehmann and Niisslein-Volhard, 1986; Schiipbach and Wieschaus, 1986) that the posterior pathway gene products contribute to, and direct assembly of, the macromolecular structures underlying polar granules. In the experiments described here, we ask if increasing the amount of one of the posteriorly localized components of the posterior pathway can affect body patterning or pole cell formation. Overexpression of osk leads to higher levels of osk mRNA, both correctly localized to the posterior pole and unlocalized. Consequently, nos is activated ectopically, causing extensive shifts in body patterning.

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several recombinant second and third chromosomes, each carrying two osk transgenes. Flies homozygous for any one of these recombinant chromosomes carry six copies of the osk gene, and are hereafter referred to as 6 x osk. Because osk is a maternal effect gene, the phenotype of the embryo is dependent upon the genotype of the mother; for simplicity we refer to embryos produced by 6 x oskmothersas6x oskembryosandfollowthisconvention for all maternal effect genes discussed. The cuticular phenotypes of 6 x osk embryos are shown in Figure 2 (note that the phenotypes are due to overexpression of osk, as described in Experimental Procedures). All pattern defects seen in 6 x oskembryos require nos. When additionally mutant for nos, 6 x oskembryos display the characteristic nos- phenotype (Lehmann and Nusslein-Volhard, 1991), a lack of abdominal segmentation (see Table 1). Hyperactivation of nos activity throughout the embryo can explain the body patterning phenotypes, as outlined in the legend to Figure 2. Figure 1. Distribution

of osk mRNA in Wild-Type

and 8 x osk Embryos

(A) Wild-type embryo. (9) 8 x oskllD embryo. Embryos are at similar preblastoderm stages and were processed for and subjected to in situ hybridization in parallel. Embryos are oriented with anterior to the left and dorsal uppermost. In the wild-type embryo osk mRNA is concentrated at the posterior pole and present at a low level throughout the embryo, as described previously (Kim-Ha et al., 1991; Ephrussi et al., 1991). In the 8x osk embryo the amount of osk mRNA localized to the posterior pole is significantly increased. In addition, there is a higher level of osk mRNA dispersed throughout the embryo.

Germ cell formation is also affected: excess posterior pole cells appear and presumptive pole cells form at an anterodorsal position. Remarkably, the latter effect is enhanced by genetically decreasing nos activity, suggesting that the action of nos may in some way deplete the pool of potential germ cell determinants. Results Increasing osk Gene Dosage Causes Body Patterning Defects The previous demonstration that fully functional copies of the osk gene could be introduced into flies by P elementmediated transformation (Kim-Ha et al., 1991; Ephrussi et al., 1991) offered a straightforward way to vary the osk gene dosage. In initial experiments we discovered that embryos laid by females carrying four extra copies of the osk gene, two on the second chromosome and two on the third, rarely hatched. These embryos had elevated levels of osk mRNA, both localized to the posterior pole and dispersed uniformly along the anteroposterior axis (Figure 1). The cuticular patterns displayed by the unhatched embryos (described below) suggest that overexpression of osk causes the posterior body patterning determinant, nos, to be activated ectopically. To address in detail the effects of increased osk gene dosage on anteroposterior body patterning, and to facilitate subsequent genetic manipulations, we constructed

Overexpression of osk Causes Ectopic Synthesis of nos Protein in 6 x osk Embryos In wild-type preblastoderm and blastoderm stage embryO8, a gradient of nos protein emanates from the posterior pole and extends a short distance anteriorly (Figure 3A). In 6 x osk embryo8 the level of nos protein is consistently increased at the posterior pole. In addition, nos protein now appears throughout much or all of the embryo (Figures 3B-3D). Thus, the amount of osk product and the level of nos protein accumulation are directly related. In addition, there appears to be a threshold level of osk required for accumulation of detectable levels of no8 protein. Although nos protein is detected only at the posterior pole of wild-type embryos, osk mRNA is present throughout wild-type embryos (as well as being highly concentrated at the posterior pole) (Kim-Ha et al., 1991; Ephrussi et al., 1991). In contrast, nos protein appears throughout 6 x osk embryos that have higher uniform levels of osk mRNA (see Figure l), indicating that the threshold value is frequently exceeded throughout much of the body. In a minor fraction of 6 x osk embryos, an anterior accumulation of nos protein is also observed, accompanied by the appearance of nos protein throughout the embryos (Figure 3D). In contrast, bicaudal embryos arising from a mutation at the Bicauda/D @CD) gene (see Figure 2 legend) have no detectable nos protein in the central regions of the body (Figure 3E). For both 6 x osk and BicD mutants the anterior accumulation of nos protein may be due to a defect in the localization of osk mRNA during oogenesis. In both 6 x osk and 6icD ovaries an intermediate in osk mRNA localization, accumulation at the anterior margin of stage 9-10 oocytes, is enhanced and prolonged relative to wild type (data not shown; Kim-Ha et al., 1991; Ephrussi et al., 1991). This defect is seen in all BicD oocytes, but only a small fraction of 6x osk oocytes. Thus the pathway for posterior localization of osk RNA appears to be close to saturation when osk is overexpressed. This anterior mislocalized osk mRNA presumably underlies the appearance of ectopic nos protein, since the proportion of embryos with anterior nos protein roughly corresponds to the fre-

oskar Directs nanos Activation 651

Figure 2. Body Patterning

and Pole Cell Formation

Phenotypes

Resulting from Overexpression

of osk

Micrographs show ventral cuticle patterns (dark-field illumination), oriented with anterior at the top. Embryos from 6 x osk mothers display a range of segmental defects. The observed phenotypes are divided into three general classes (see Table 1 for the distribution of embryos among the different classes). (A) The first and least frequent class includes those embryos with apparently wild-type cuticles. (B and C) Embryos of the second class retain normal anteroposterior polarity but show progressive loss of anterior pattern elements. Within this class the least severe examples have only reduced head structures, while the most severe lack all head and thoracic structures and retain as few as four abdominal segments. (D and E) In the third and most frequent class are bicaudal embryos in which head, thoracic, and anterior abdominal segments are replaced by a mirror image duplication of posterior abdominal segments and telson. 6 x osk bicaudal embryos may be symmetric or asymmetric with as many as three abdominal segments in the anterior opposing those in the posterior. The appearance of bicaudal embryos invites comparison with the well-characterized example of 6icD (Mohler and Wieschaus, 1966; Suter et al., 1969; Wharton and Struhl, 1969). In BicD embryos, nos is activated not only at the posterior but also at the anterior pole. Although superficially similar, the cuticular phenotypes of 6x osk and 6icD embryos differ significantly; BicD embryos typically have four or more duplicated abdominal segments, while 6 x osk embryos typically have three or fewer duplicated abdominal segments, As described below, the two types of bicaudal embryos arise in different ways. Cur interpretation of how overexpression of oskalters body patterning follows from a wealth of recent work describing the role of the hb morphogen rn specifying posterior pattern formation (Hijlskamp et al., 1990; Eldon and Pirrotta, 1991; Kraut and Levine, 199lb, 1991a; Struhl et al., 1992) and describing the action of nos in repressing translation of both hb and bed mRNAs (Wharton and Struhl, 1991). When nos activity is restricted to the posterior pole, it has no effect on anteriorly localized bed mRNA and represses translation of hb mRNA only in the posterior. As nos activity appears at a low level throughout the embryo, there are two effects: expression of bed is reduced, leading to a loss of head structures, and early expression of hb is also reduced, directly by translational repression of maternal hb mRNA and indirectly by lowering bed-dependent zygotic hb transcription. Changing the profile of the early hb morphogen gradient expands posterior positional values at the expense of anterior values. Further increases in nos activity enhance these effects. When the level of nos activity is sufficient to abolish early hb expression, the terminal system alone contributes body polarity (Ntisslein-Volhard et al., 1967; St Johnston and Niisslein-Volhard, 1992) forming bicaudal embryos. Notably, 6 x osk bicaudal embryos have fewer abdominal segments than do BicD embryos. In BicD mutants ectopic nos is restricted to the anterior pole; consequently, some hb protein remains in the center of the embryo and contributes to the body pattern (Wharton and Struhl, 1969). In 6 x oskembryos the more uniform activation of nos eliminates this central domain of hb (data not shown), deleting the more anterior abdominal segments present in 8icD embryos. Indeed, the phenotype of embryos from hb-bet mothers (Hiilskamp et al., 1990) is identical to that of 6x osk bicaudal embryos. The expression patterns of hb, knirps, Kruppel, and even-skipped(see Niisslein-Volhard (1991)) are all altered in 6 x oskembryos in a manner consistent with this interpretation. First, moderate losses of anterior pattern elements are compensated by expansion of posterior pattern elements. Second, in embryos with more severe anterior deletions, posterior pattern elements are duplicated and inverted in the anterior (data not shown).

quency of oocytes with the osk mRNA localization defect. Note that the fraction of 6 x oskembryos with ectopic nos protein at the anterior pole is much too low to account for the bicaudal phenotype (Figure 3 legend), although mislocalization of nos protein may contribute to the observed phenotypes. Increasing ask Gene Dosage Affects Pole Cell Formation Overexpression of osk clearly stimulates the pathway

leading to localization and activation of the posterior body patterning determinant nos. Since this pathway is also responsible for pole cell formation, we would like to know if elevated levels of osk also stimulate that process. Inspection of 6x osk embryos at the cellular blastoderm stage suggests that the number of posterior pole cells is increased relative to wild type. To obtain a quantitative measure of this effect, we counted pole cells in gastrulati iiig embryos. We find an average of 35.5 pole cells in wild-type embryos in contrast with an average of 49.2 in

Cdl

852

Figure 4. Ectopic Pole Cells in 6 x osk Embryos All embryos have been stained immunohistochemically to detect nos protein. Note that nos protein is specifically localized to pole cells in cellularized embryos and never appears at any position other than the posterior pole cells in wild-type cellular blastoderm stage embryos (data not shown). (A) 6 x osk embryo at cellular blastoderm stage. nos protein is specifically localized to the posterior pole cells. In addition, at one position along the anterodorsal surface of the embryo a few cells also contain nos protein. These cells (shown at greater magnification in the inset) are round and appear identical to the posterior pole cells, unlike the adjacent elongate cells. The ectopic pole cells appear in about half of all 6 x osklllA embryos. Thus it seems that 6 x os&lllA embryos are just at the threshold of forming these cells. Indeed, not all 6x osk stocks form ectopic pole cells. (B) 6 x ask; nos. embryo at cellular blastoderm stage. The presumptive pole cells are more pronounced (inset), and appear in about half of all 6 x osk; nos. embryos (our standard second chromosome 6 x osk stock by itself fails to make ectopic pole cells). Note that the nos allele used (no&‘) makes the nos antigen. (C) 6 x ask; no.s embryo at gastrulation stage. The ectopic pole cells persist and continue to stain intensely with nos antisera. Figure 3. Distribution Embryos

of nos Protein in Wild-Type,

6x osk, and BicD

All embryos are at similar preblastoderm stages and were processed for and subjected to immunohistochemistry in parallel. (A) Wild-type embryo. nos protein is highly concentrated at the posterior pole. A gradient of nos protein appears to emanate from the zone of concentration and extends anteriorly. (B-D) 6 x osk embryos. nos protein now appears at high levels in a much larger and less precisely defined posterior region. In addition, nos protein is present to varying degrees throughout these embryos. For most embryos the pattern seems to reflect a low level of uniform nos protein accumulation, coupled with a high level of expression at the posterior pole. (D) In a small fraction of 6 x osk embryos there is

specific anterior accumulation of nos protein, in addition to the increased levels throughout and at the posterior pole. This pattern is seen for about 15% of 6 x osk embryos at the appropriate stage. Note that 73% of embryos from the same 6 x osk stock display the bicaudal phenotype. (E) 8icD embryo. The normal posterior distribution of nos protein is present. In addition, there is a diffuse anterior zone of nos protein. There is no detectable staining in the central region of the embryo.

oskar Directs nanos Activation 653

and Pole Cell Formation

6 x osk embryos, a statistically significant difference (t = 3.65, P = 0.002; see Experimental Procedures). Thus, overexpression of osk increases the number of posterior pole cells. Furthermore, hints of ectopic pole cell formation emerged from examination of nos protein distribution in 6 x oskembryos. In some of the cellularized embryos, nos protein is noticeably concentrated in a few morphologically distinct cells positioned dorsally and just anterior to where the cephalic fold would soon form. At late blastoderm stage the cells surrounding the periphery of the embryo are elongate, while these unusual cells are round and resemble pole cells (Figure 4A; both cell types are readily visible in the inset). However, these cells could not be detected after the onset of gastrulation. Fortuitously, we discovered that in 6 x osk; nos- embryos the appearance of these cells was enhanced (Figure 46), and they persisted into gastrulation (Figure 4C). All of the round cells contain a high level of nos protein, and some also contain a high level of vas protein (data not shown). vas protein is normally present throughout the embryo but concentrated in the pole cells (Hay et al., 1966; Lasko and Ashburner, 1990). We tentatively refer to the novel round cells as ec-

Table I, The 6 x osk Cuticular

Phenotype Cuticular

genotype’

Posterior Deletions

+; 6 x osk vas; + vas; 6x osk

0 100 100

osk osk

Maternal

v/s-; + vls; 6x

tuct; + tub;

6x

in Wild-Type Phenotype

topic pole cells, although proof would require a demonstration of their function in a transplantation assay. The enhanced formation of ectopic pole cells in a nos mutant background is a curious and unexpected result, since there have been no previous indications that nos is in any way involved in pole cell formation. This observation led us to ask if nos normally represses formation of posterior pole cells in wild-type embryos. Indeed, we find an average of 44.1 pole cells in nos- mutants, which is significantly higherthan theaverageof 35.5 in wildtypeembryos (t = 2.73; P = 0.014; see Experimental Procedures). Presumably, the same mechanism responsible for the increased number of posterior pole cells in nos mutants underlies enhancement of ectopic pole cells in 6 x ask; nosembryos. Requirements for Posterior Group Genes in Ectopic nos Activation and Ectopic Pole Cell Formation To determine which of the posterior group genes are required to convert the overexpression of osk to ectopic activation of nos and pole cell formation, we have introduced mutations in these genes into 6 x osk stocks and exam-

and Posterior Group Mutant Backgrounds

(o/o)” Anterior Defects

Bicaudal

N

1 0 0

34 0 0

66 0 0

155 26 72

100 100

0 0

0 0

0 0

160 36

57 67

43 33

0 0

0 0

176 125

Wild Type

stau-; + stau-; 6 x osk

1ooE 16’

0 0

0 22

0 62

103 412

spir; + spir-; 6 x osk

100 10

0 1

0 47

0 42

164 92

cape-; + capu- 6 x osk

100 5

0 3

0 56

0 35

259 107

0 100 99

1 0 0

26 0 1

73 0 0

22% 137 146

+; pum6 x ask; porn-

97 5

3 27

0 65

0 4

296 229

4x 6x

0 10

53 67

29 9

17 14

215 467

6 x osk; + +; nos 6 x osk; nos

osk; + osk; oslr

* Standard Drosophila genetics conventions are used in describing genotypes, with the relevant second and third chromosome genotypes presented in order and separated by a semicolon. Two different 6 x osk stocks were used in these experiments. For the posterior group mutations on the second chromosome, 6 x osklllA was used, and ior the mutations on the third chromosome 6 x oaklID was used. Each of these 6 x osk stocks is presented above the appropriate group of mutants, and highlighted in bold type. b Cuticular phenotypes were scored as follows. Posterior deletions: embryos lacking one or more abdominal segments but otherwise wild type. Wild type: embryos with no apparent cuticular defects. Anterior defects: embryos that display normal anteroposterior polarity but show progressive loss of anterior pattern elements. Bicaudal: embryos with duplicated posterior structures inverted in the anterior. Embryos with poorly developed cuticles were not counted. The spir; 6 x osk and cepu-; 6 x osk genotypes produce more than the normal amount of poorly developed cuticles. This may contribute lo the change in the relative distribution of cuticles between the last two columns, as compared with 6 x osk alone. ’ The steu gene is required for both anterior and posterior body patterning. Consequently, the steu cuticles in the posterior deletion category also display anterior defects.

phenotype. For example, vas-; 6 x osk embryos exhibit a typical vas- cuticular phenotype: deletion of most abdominal segments (Schiipbach and Wieschaus, 1966). Moreover, they fail to form any pole cells. Similarly, the phenotypes of vls-; 6x osk and tu&; 6 x osk embryos are essentially the same as those of the v/s- and tuck mutants, respectively (Boswell and Mahowald, 1965; Schi.ipbach and Wieschaus, 1966). Those genes thought to be required only for localization of nos (Manseau and Schtipbach, 1969; Lehmann and Ntisslein-Volhard, 1991) behave differently in their effects on the 6 x osk phenotype. Mutations in stau, spir, or capu alter the 6x osk cuticle phenotypes by adding a class of embryos with abdominal deletions typical of posterior group mutants (Table 1). This class presumably corresponds to the 6 x osk embryos that have the lowest levels of osk mRNA, including those in the wild-type class and the weakest members of the anterior deletions class. Surprisingly, some stau-; 6 x osk, spir; 6 x osk, and capu-; 6x osk embryos retain normal polarity, implying an uneven distribution of nos protein. We therefore examined nos protein distribution in these embryos. Although no localized nos protein was found in stau-, capu-, or spir embryos, nos protein does accumulate at one or both poles in some stau-; 6 x osk, spir; 6 x osk, and capu-; 6x osk embryos. When nos protein is localized to only one pole, we infer that it is the posterior pole, since cuticle preparations show that embryos with normal polarity are always in the normal orientation in their vitelline membranes and never inverted (data not shown). (Such inversions occur when stau, capu, or spir mutations are combined with a BicD mutation [Manseau and Schiipbach, 1989; Lehmann and Niisslein-Volhard, 19911.) Therefore, higher levels of oskcan effectively circumvent the defects in the posterior localization pathway associated with these particular mutations. Moreover, pole cells form in some stau-; 6 x osk, spir; 6 x osk, and capu-; 6 x oskembryos. These pole cells can appear either at the posterior pole, at the anterior ectopic site, at a novel posterior ectopic site, or at a combination of the above (Figure 5 and Table 2). The final posterior group gene to be tested for its role in expression of the 6x osk phenotype is pum. Curiously, when osk is overexpressed in apum mutant background, many embryos display wild-type cuticular patterns. Some hatch to produce viable adults. Thus, overexpression of osk can rescue the pum mutant phenotype; in turn, the pum mutation reduces the severity of the 6 x osk phenotype (Table 1). It seems, then, that thepum mutation coun-

C ,‘”

i ,’.* ‘1”’ Figure 5. Pole Cells in 6x

ask; stau- and 6x

ask; spir

Embryos

All embryos have been stained immunohistochemically to detect nos protein. (A and B) 6 x ask; stau-. Pole cells form at the wild-type position at the posterior pole in the embryo shown in (A). Ectopic anterior pole cells are also found in these mutants, and their presence is independent of posterior pole cells (B). Note that pole cells are never found in singly mutant stau- embryos. (C) 6 x osk; spir embryo. In this genotype ectopic pole cells have been found at a posterior position similar to the anterodorsal location where the ectopic pole cells typically form. Ectopic pole cells are seen at both locations in this embryo, which is presented as a composite of micrographs of two different focal planes in order to show the pole cells more clearly. Similar results are obtained for 6 x osk; capu- embryos (Table 2).

ined the phenotypes (Table 1). All posterior group genes thought to function in both activation and localization of the posterior determinant (Lehmann and NiissleinVolhard, 1991) are necessary for expression of the 6 x osk

Table 2. Sites of Pole Cell Formation

Maternal

Genotype

in 6x

osk Embryos

Also Defective

in stau, sp[f, or capu

c)n--

s&u; 6x osklllA spir; 6 x osklllA capu; 6 x osklllA n Total number of appropriately

IV 9 5

staged embryos

1 3 2 scored. All numbers

2

in other columns

33 25 6

6

refer to embryos,

not percentages.

oskar Directs nanos Activation 855

and Pole Cell Formation

Figure 6. A Mutation in pumDoes Not Affect the Pattern of nos Protein in 6 x osk Embryos Both embryos are at similar preblastoderm stages and were processed for and subjected to immunohistochemistry in parallel. (A) 6x osk embryo. (B) 6 x ask; pum- embryo. Both genotypes display high levels of nos protein at the posterior pole and throughout the embryo. Although there is some variability in the pattern of nos protein in 6 x osk embryos (see Figure 3), we found no consistent change in the distribution or reduction in the amount of nos protein in the 6 x osk; pum- mutants. If anything, addition of the pum mutation to the 6 x osk stock caused a slight increase in the amount of nos protein.

teracts hyperactivation of nos. Comparison of the distribution of nos protein in 6 x osk and 6 x osk; pum- embryos reveals no obvious differences (Figure 6), suggesting that pum acts not at the level of nos protein synthesis, stability, or movement, but rather by promoting activation or preventing deactivation of nos protein. We have not seen ectopic pole cell formation in the 6 x osk; pum- embryos. Because the 6 x osk stock used with third chromosome mutations (including both pum and nos) does not by itself form ectopic pole cells (see Figure 4 legend), it is difficult to assess the effect of the pum mutation on this phenomenon, except to say that whatever effect there may be is much less extreme than observed for the nos mutation (as described above).

Discussion A Threshold of osk Is Required for Production of Body Patterning and Germline Determinants The most striking effect of overexpressing osk is that both body patterning and germ cell formation are affected. Activities of both the nos morphogen and the germ cell determinant are increased at the posterior pole. Moreover, both determinants now act elsewhere in the embryo: the nos body patterning activity is found spread throughout the embryo, corresponding to the distribution of nos protein; and a novel cell type, which appears to be equivalent to germline pole cells, appears at an anterodorsal position.

An understanding of how the determinants are produced ectopically may help explain how osk acts in the posterior pathway. In principle, the ectopic appearance of determinants could arise from local activation or by diffusion from a source at the posterior pole. Patterns of nos protein accumulation show that local activation does occur. In particular, a small fraction of embryos display a discrete anterior accumulation of nos protein. Moreover, in many embryos with largely uniform nos protein, the nos gradient from the posterior extends only a short distance anteriorly, suggesting that it is not the source for all nos protein. Thus accumulation of nos protein can occur locally, and the appearance of ectopic pole cells at the anterodorsal position suggests that the germline determinant can also be produced away from the posterior pole. Normally osk mRNA is present throughout the embryo, as well as being concentrated at the posterior pole. Nevertheless, nos protein is strictly confined to the posterior pole, as is the activity directing germ cell formation. Since we find that an increase in the level of osk throughout the embryo leads to local accumulation of nos protein, it seems that when the level of osk gene product exceeds a critical threshold, the posterior pathway is initiated. This possibility was previously suggested by analysis of BicD mutants, in which the posterior patterning activity is ectopitally localized to the anterior pole of the embyro (Lehmann and Niisslein-Volhard, 1986). In these mutants anterior accumulation of osk mRNA during oogenesis is enhanced and prolonged relative to wild type (Kim-Ha et al., 1991; Ephrussi et al., 1991). Although there is no proof that the effect on osk was solely responsible for the body patterning phenotype, our work now supports the notion that a critical threshold level of oskaccumulated at the anterior pole of these mutants. The BicD mutants highlight a feature of the posterior pathway related to activation threshholds. Although nos is ectopically activated in BicD mutants, ectopic pole cells are not observed (Mohler and Wieschaus, 1986). Similarly, there are alleles of several posterior group genes that specifically interfere with germ cell formation, but retain normal body patterning activity (Boswell and Mahowald, 1985; Lehmann and Niisslein-Volhard, 1986, 1991). This feature could reflect differences in the amounts of precursors required for activation of the different determinants (Lehmann and Niisslein-Volhard, 1986). The effects of overexpression of osk suggest the same conclusion. Almost all 6 x oskembryos display body patterning defects, with significantly enhanced levels of nos protein along part or all of the anteroposterior body axis. In contrast, formation of ectopic pole cells is weak and not seen in all of the 6 x osk embryos. Thus the posterior pathway may need to be activated to a much greater level to induce germ cell formation. If so, the wild-type embryo may face a problem in ensuring that the posterior pathway is sufficiently active to direct pole cell formation, yet not so active as to generate too much nos activity. Indeed, a P-fold increase in osk leads to body patterning defects (Table l), indicating that osk is normally close to a detrimental level at which too much nos appears. How are slight fluctuations in osk expression prevented from interfering with body patterning?

It is tempting to speculate that a protective mechanism exists to deactivate the low levels nos produced inappropriately in such situations, either as it is activated ectopicaky or as it diffuses away from the posterior pole. Such a system could be further buffered by a function involved in maintaining nos activity. Perhaps this is the role played by pumilio (see below). Roles of the Posterior Group Genes in nos Activation and Germ Cell Formation The posterior group genes have been divided into functionally distinct classes based on their effects on the nos morphogen. One class, consisting of osk, vas, v/s, and tud, is required to produce nos activity (Boswell and Mahowald, 1985; Mohler and Wieschaus, 1988; Lehmann and Niisslein-Volhard, 1988, 1991). Another class, including spir, capu, and stau, appears to be necessary only for posterior localization of nos (Manseau and Schiipbach, 1989; Lehmann and Niisslein-Volhard, 1991). Our experiments support this distinction and allow us to ask if the posterior group mutants can be placed into the same classes on the basis of their effects on pole ceil formation. Mutations in vas, v/s, and tudeliminate nos activity and pole cell formation, independent of osk gene dosage. The results of combining 8 x osk with spir, capu, or stau mutants are more complex. The spir, capu, orstau mutants do not interfere with expression of the 8 x osk body patterning phenotype. This result is consistent with roles for these genes in localization of nos. However, none of these double mutants is completely defective in posterior localization, as shown by the concentration of nos protein at the posterior pole of some embryos. Furthermore, posterior pole cells form in some stau-; 8 x osk and spir; 8 x osk embryos. The capu and spir mutations used here still retain partial function (Manseau and Schupbach, 1989) and it may be that they can support a very low level of osk mRNA localization, normally not detectable but significant when osk is overexpressed. The failure of the stau mutation to abolish localization may be more interesting. The stau allele used, stauD3, has a significant deletion (- 250 bp) near the 5’ end of the coding region and makes no detectable protein (St Johnston et al., 1991); thus it should probably be viewed as a null mutation. Moreover, there is no detectable posterior localization of osk mRNA or osk protein in the stau-; 8x osk embryos (data not shown). Nevertheless, nos protein is localized to the posterior pole, and posterior pole cells are found in some embryos. Thus there may be redundancy in posterior localization, and failure at one level could be partially restored at a different level. Role of pum in Posterior Body Patterning A dramatic effect of overexpression of osk is rescue of the pum phenotype. Typically, pum mutants form only two abdominal segments. When osk is overexpressed in a pum mutant background, a substantial fraction of the embryos display normal segmentation. Furthermore, some embryos hatch and are viable. pum apparently acts only in the body patterning part of the posterior pathway (Lehmann and Niisslein-Volhard, 1988), and we have now

shown that it does so in a manner independent of accumulation and distribution of nos protein. Thus the role of pum must be in ensuring that nos protein is present in an active form. To accomplish this pum could posttranslationally activate nos protein or stabilize or prevent deactivation of nos protein (Macdonald, 1992). Apparently the excess nos protein provided by overexpression of oskmakespum partially redundant. We favor the notion thatpum acts to stabilize or prevent deactivation of an activated form of nos protein. Deactivation could normally occur as nos protein moves away from the posterior pole early in embryogenesis. This interpretation is fully consistent with the results of cytoplasmic transplantation experiments, in which pum mutant embryos have been found to contain fully active posterior patterning activity at their posterior poles (Lehmann and Niisslein-Volhard, 1991). Pole Cell FormationDo the Two Branches of the Common Posterior Pathway Compete for a Limiting Precursot? Perhaps the most surprising consequence of the overexpression of osk is the ectopic appearance of presumptive pole cells. The only previous demonstration of pole cell formation at a position other than the posterior pole was in experiments in which polar plasm was transplanted to anterior or ventral positions in recipient embryos (lllmensee and Mahowald, 1974,1978; Niki, 1988). 8 x osk ectopic pole cells form almost exclusively at a particular anterodorsal position, raising the possibility that specific spatial cues constrain or influence this process. It will be of interest to determine what features permit formation of pole cells at this site. One previously unsuspected aspect of the posterior pathway is suggested by enhancement of ectopic pole cell formation in nos mutants. There are several possible explanations for this phenomenon. First, ectopic pole cell formation may be more productive in body regions specified as anterior. In 8 x oskembryos the entire embryo has posterior positional values, while in 8 x osk; nos- embryos the region where the ectopic pole cells form now has an anterior value. We do not favor this explanation since it cannot account for the formation of ectopic pole cells in many stau-; 8 x osk, spit-; 8 x osk, and capu; 8 x osk embryos, which retain posterior values anteriorly. A second possibility is that nos may down-regulate a factor required for pole cell formation. If so, this would likely occur at the level of translation, much as nos regulates expression of bed and hunchback (hb) (Wharton and Struhl, 1991). Finally, there may be competition between the two branches of the posterior pathway, perhaps for a shared and limiting precursor. When the posterior body patterning determinant is impaired, it no longer withdraws precursors from the common pool, leaving more available for pole cell formation. The failure of apum mutant to enhance ectopic pole cell formation does not argue against this last model. While the pum mutant does reduce nos activity, there is no reason to believe that this effect would be exerted prior to the utilization of the shared precursor by nos. Further analysis of the posterior pathway will likely reveal which possibility is correct.

oskar Directs nanos Activation 057

and Pole Cell Formation

Experimental Procedures Fly Stocks w”‘~ flies were used as “‘wild-type” controls and as recipients for P element transformation. For immunohistochemistry and in situ hybridization analysis in maternal mutant backgrounds, the following homozygous mutant stocks were used, in combination with 6 x os&llD (for third chromosome mutants; 6 x osk stocks are described in the following section) or 6 x osklllA (for second chromosome mutants) as appropriate: capuVcapu2; spir’lspir’; stauVsta3: vasVvas’; vls’lvls’; WI tuti; osPlosP, osk2loskz; pum’31pum’3; no.Flnos”. BicD mutant mothers were BicD’IBicD2. All mutants are described in Lindsley and Zimm (1992).

Constructlon and Characterlxatlon of Stocks Carrying Six Copies of osk We previously described transformantflies that rescued the osk maternal effect phenotype (Kim-Ha et al., 1991). After characterization of additional independent transformant lines we found that of five total, one did not rescue. To improve the likelihood that any given transformant would provide full osk function, we used a different set of 20 fly lines transformed with a 8 kb osk genomic DNA fragment (P[osklOB-I] to P[osklO&201). Each of the 10 second chromosome insertions tested rescued oak function. Several recombinant second or third chromosomes, each carrying two independent and homozygous viable P[osklOB] transgenes, were constructed by standard methods. The P[osklOB] transgenes included in the stocks are as follows: 6 x oskllA, 16 and 19; 6x oskllt3, 16 and 17; 6x oskllC, 6 and 17; 6x oskllD, 6and 12; 6x osklllA, 2and9; 6x osklllB, 2and3; 6x osklllC, 3 and 4; 6x osklllD, 3 and 10. Flies homozygous for one such chromosome were collected and placed into a small cage with an apple juice plate at 25“C. Egg collections and cuticle preparations were done as described by Wieschaus and Ni.isslein-Volhard (1986), with a few modifications. Vitelline membranes were removed by transferring the embryos into a scintillation vial containing 9 ml of methanol, 1 ml of 0.5 M EGTA, and 10 ml of heptane and vortexing intermittently over a 5-10 min period. Devitellinized embryos, which settle in the aqueous phase, were rinsed in 0.1% Triton X-100, transferred into Hoyers:lactic acid (5:3), mounted on slides, and heated at about 60°C overnight. In some cases the embryos were extremely fragile, and many cuticles did not survive the preparation. To reduce this problem, the embryos were fixed prior to removing the vitelline membranes. Fixing was in a I:1 mixture of heptane: 4% formaldehyde, 100 m M HEPES (pH 6.8), 1 m M EGTA, 2 m M MgS04 for 20 min. Two types of evidence indicate that the 6 x osk cuticular phenotypes observed are due to overexpression of osk. First, most stocks produce

Table 3. Effects of osk Overexpression

on Embryonic Cuticular

Maternal Genotype*

Wild Type

w71’B

100 17 11 7

6x 6x 6x 6 x 6 x 6x 6x 6x

oskllA oskllB oskllC

oskllD osklllA

1 1

osklllB osklllC osklllD”

6 4 30

similar proportions of embryos in different phenotypic classes (Table 3). Thus, effects due to gene disruptions from insertion of the transgenes are negligible. The modest phenotypic differences among the various stocks presumably reflect slightly different levels of transgene expression. One exceptional stock displays less extreme phenotypes and includes a transgene that may be expressed only poorly. Second, the mutant phenotypes are not due to some other effect of the transformed DNA, as demonstrated by placing one of the 6 x osk stocks into a homozygous ask- background. These 4x osk flies, in which all four osk copies are transgenes, were compared with 4 x osk flies, of which only two copies were transgenes. The embryonic phenotypes resulting from the 4 x osk transgene flies were no more severe than those from the 2 x osk endogenous; 2 x osk transgene flies (Table 1). In fact, the former were consistently less severe, suggesting that the transgenes may be expressed somewhat irregularly, or that the transgene transcripts may be localized less consistently than those of the endogenous genes. Analysis of 6 x osk stocks in various mutant backgrounds was done essentially as described above for the 6 x osk stocks alone. In some cases the embryos for cuticle preparations were so fragile that they did not survive devitellinization even after formaldehyde fixation. These were fixed and mounted in their vitelline membranes.

RNA Analysis In situ hybridization to whole mount ovaries and embryos followed the procedures of Tautz and Pfeifle (1989), using digoxigenin dUTPlabeled DNA probes prepared by random priming according to the protocols provided with the Genius labeling kit (Boehringer Mannheim). The nos probe was a 780 bp Sty1 restriction fragment (nucleotides 61 l-1391 of Wang and Lehmann [1991]) of a cDNA obtained by screening an ovarian cDNA library with an oligonucleotide specific for the nos gene (Wang and Lehmann, 1991). The osk probe was a 1.6 kb restriction fragment extending from a Hindlll site to a Sty1 site (nucleotides 884-2471 of Kim-Ha et al. [1991]).

Antlbodles and lmmunohistochemlstry lmmunohistochemistry was performed as described previously (Macdonald and Struhl, 1986; Macdonald et al., 1991), using secondary antibodies coupled to horseradish peroxidase (Jackson Immunoresearch Laboratories). Antisera specific for hb, Kriippel, knirps, and even-skipped proteins were prepared in rats and have been described previously (Struhl et al., 1989; Macdonald, 1990; Wharton and Struhl, 1989). The antisera directed against vas and nos proteins were prs pared in rats using bacterial fusion proteins prepared as described previously (Macdonald, 1992). The vas expression construct (Wharton and Struhl, 1989) was a gift from Robin Wharton. The nos expression construct consists of a Banll-EcoRI fragment (nucleotides 388 to

Body Pattern

Phenotype

(o/o)” Anterior 34 32 26 26 34 20 17 61

Defects

Bicaudal

N

49 57 67 73 66 67 79 9

137 401 289 335 220 155 18 47 205

a All 6 x osk stocks are homozygous for a recombinant chromosome (II or Ill) bearing two osk transgenes (Experimental Procedures). 6 x oskllD and 6 x osklllA (bold) are used for all the experiments described here. ’ Cuticular phenotypes were scored as follows. Wild type: embryos with no apparent cuticular defects. Anterior defects: embryos that display normal anteroposterior polarity but show progressive loss of anterior pattern elements. Bicaudal: embryos with duplicated posterior structures inverted in the anterior. Embryos with poorly developed cuticles were not counted. These were seen more often than usual in several lines (IIIA, Ill& IIIC), and we suspect that severity of the phenotype may be underestimated. ’ 6 x osklllD shows an uncharacteristic phenotypic distribution: a majority of embryos exhibit only anterior defects. Of the 6 x osk lines analyzed, this stock alone includes osk transgene P[osklO&lO], which we suspect to be poorly expressed.

Cdl 858

-2150),from (Rosenberg

thecDNAdescribedabove, inserted inframeintopET3a et al., 1987) and was a gift from Robin Wharton.

Pole Ceil Counts Gastrulating embryos were collected, fixed, and stained with antibodies against nos protein as described above. Embryos were viewed on a video monitor. Pole cells were counted by taking optical sections through the embryo and marking all pole cells on a transparent plastic sheet taped to the video screen. To avoid bias, embryos of different genotypes were analyzed at random. One person placed the specimens on the microscope, and another person performed the counts at the video monitor with no knowledge of the genotype. Ten embryos of each genotype were counted and the counts averaged. Comparisons were made using one-tailed t tests.

Acknowledgments We thank Robin Wharton for expression constructs, Lisa Jack for help with statistical analysis, Bruce Baker, Madison Kilpatrick, Jeongsil Kim-Ha, Sandy Luk, Carol Rohl, Gary Struhl, Philippa Webster, and Robin Wharton for critical reviews of manuscript, and Lex Bunten for help with preparation of the manuscript. This work was supported by a David and Lucille Packard Fellowship and in part by a grant from the National Institutesof Health (GM42612). P. M. M. is a Pew Scholar in the Biomedical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adveftisemenf” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

July 6, 1992; revised July 28, 1992.

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