Nanos downregulates transcription and modulates CTD phosphorylation

in the soma of early Drosophila embryos

Girish Deshpande, Gretchen Calhoun, Timothy M. Jinks1,Alexandros D. Polydorides2, Paul Schedl*

Department of Molecular Biology, Princeton University, Princeton, NJ 08540, USA

Received 3 August 2004; received in revised form 17 December 2004; accepted 17 December 2004

Available online 26 January 2005

Abstract

nanos (nos) specifies posterior development in the Drosophila embryo by repressing the translation of maternal hb mRNA. In addition to

this somatic function, nos is required in the germline progenitors, the pole cells, to establish transcriptional quiescence. We have previously

reported that nos is required to keep the Sex-lethal establishment promoter, Sxl-Pe, off in the germline of both sexes. We show here that nos

also functions to repress Sxl-Pe activity in the surrounding soma. Sxl-Pe is inappropriately activated in the soma of male embryos from nos

mothers, while Sxl-Pe can be repressed in female embryos by ectopic Nos protein. nos appears to play a global role in repressing transcription

in the soma as the effects of nos on promoter activity are correlated with changes in the phosphorylation status of the carboxy terminal

domain (CTD) repeats of the large RNA polymerase II subunit. Finally, we present evidence indicating that the suppression of transcription

in the soma by Nos protein is important for normal embryonic development.

q 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: nanos; C-terminal domain (CTD); CTD phosphorylation; Sex-lethal; Transcriptional repression

1. Introduction

Abdominal fate in the Drosophila embryo is specified by

nanos (nos) mRNA localized in the posterior pole plasm

(St Johnston, 1993). Translation of this mRNA generates a

Nos gradient that extends anteriorly (Gavis and Lehmann,

1992). The Nos gradient represses the translation of

uniformly distributed maternal Hunchback (Hb) mRNA

(Wharton and Struhl, 1991; Wang and Lehmann, 1991).

This posterior repression, together with the transcriptional

activation of the hb gene by Bicoid in the anterior, generates

a Hb gradient at the anterior (Struhl et al., 1992; Gavis and

Lehmann, 1992). Translational repression by Nos requires

Pumilio (Pum), and it is thought that Pum interacts with Nos

0925-4773/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.mod.2004.12.009

* Corresponding author. Tel.: C1 609 258 4979; fax: C1 609 258 1028.

E-mail address: pschedl@molbio.princeton.edu (P. Schedl).1 Present address: Division of Developmental Neurobiology, National

Institute for Medical Research, Mill Hill, London NW7 1AA, UK.2 Present address: Laboratory of Molecular Neuro-oncology, Rockefeller

University, New York, NY, USA.

Response Elements (NREs) in the hb mRNA 3 0UTR

(Murata and Wharton, 1995). In the absence of either Nos

or Pum, Hb is translated from maternal mRNA in the

posterior and blocks abdominal segmentation.

In addition to its role in the soma, nos is required for the

proper development of the germline (Kobayashi et al., 1996;

Forbes and Lehmann, 1998). Pole cells in embryos from nos

mutant mothers (referred to as either nosmK embryos or pole

cells) are not correctly determined. Unlike wild type, newly

formed nosmK pole cells fail to downregulate transcription

and attenuate the cell cycle (Asaoka et al., 1998, 1999;

Deshpande et al., 1999). Later in embryogenesis they

exhibit a range of migration defects and fail to associate

with the somatic gonadal precursor cells.

One gene that is inappropriately expressed in nosmK pole

cells is Sex-lethal (Sxl) (Deshpande et al., 1999). Sxl

controls somatic sexual development, and is switched on in

females but not males (Cline and Meyer, 1996). During

most of development on/off regulation is post-transcrip-

tional; however, when sexual identity is determined Sxl is

regulated at the transcriptional level. Sexual identity is

Mechanisms of Development 122 (2005) 645–657

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G. Deshpande et al. / Mechanisms of Development 122 (2005) 645–657646

chosen at the blastoderm stage by counting the

X chromosome to autosome ratio. Counting depends upon

the expression of X-linked numerators at a level pro-

portional to their dose. The known numerators include the

transcription factors scute (sc), sisterless-a (sis-a) and runt

(rt), and the JAK/STAT ligand, unpaired (upd) (Cline and

Meyer, 1996). In 2X/2A (female) nuclei, the concentration

of numerators is sufficient to activate the Sxl-Pe establish-

ment promoter (Keyes et al., 1992). In contrast, in 1X/2A

nuclei (male), these positive factors are unable to overcome

the negative effects of autosomal denominators and Sxl-Pe

remains off. The Sxl proteins produced by the Sxl-Pe

transcripts in 2X/2A embryos activate the Sxl autoregula-

tory feedback loop by directing the female splicing of the

first transcripts from the Sxl-Pm maintenance promoter.

Translation of these female spliced Sxl-Pm mRNAs

produces Sxl proteins which autoregulate their own

expression and ensure a commitment to female identity

during the remainder of development. In males, the Sxl-Pm

pre-mRNAs are spliced in the non-productive default

pattern ensuring a commitment to male identity. While Sxl

is activated in XX soma at the blastoderm stage, it is not

turned on in the germline until much later in development

(Horabin et al., 1995). Moreover, though germline acti-

vation is sex specific, the mechanism is distinct from that in

the soma (Cline and Meyer, 1996). In fact, the available

evidence suggests that Sxl-Pe is not active in the germline.

Consequently, it is surprising that Sxl-Pe is turned on in

nosmK pole cells at the blastoderm stage in both sexes.

The ectopic activation of Sxl-Pe in nosmK pole cells

raises the question of what role, if any, the nos gene plays in

somatic sex-determination and more generally in regulating

somatic transcription. We now show that nos functions to

repress Sxl-Pe activity in the soma and that the Sxl-Pe

promoter is inappropriately turned on in the somatic 1X/2A

nuclei of nosmK embryos while it is upregulated in 2X/2A

nosmK embryos. The effects of nos on Sxl-Pe appear to be

due to a general upregulation in RNA polymerase II activity

in nosmK embryos. Transcription by RNA polymerase II is

correlated with the phosphorylation of serine residues ser2

and ser5 in the 7 amino acid (aa) repeats in the CTD domain

of the largest subunit. These two serines are thought to be

phosphorylated sequentially in the transcriptional cycle

(Dahmus, l996; Komarnitsky et al., 2000). Ser5 phos-

phorylation occurs first and is correlated with promoter

clearance, while ser2 phosphorylation occurs after clearance

and is correlated with elongation. Studies in Caenorhabditis

elegans indicate that high levels of phospho-ser2 and ser5

are present in transcriptionally active somatic nuclei. In

contrast, in quiescent germline nuclei there is little phospho-

ser2, and although polymerase with phospho-ser5 is

detected, there is much less than in the soma. In pie-1

mutants, which are unable to silence germ cell transcription,

the levels of phospho-ser2 and ser5 approach that in somatic

nuclei (Mello et al., 1996; Seydoux et al., 1996; Seydoux

and Dunn, 1997). We show here that ser2 and ser5

phosphorylation is upregulated in the germline and soma

of nosmK embryos, while both modifications can be

downregulated in the soma by ectopic Nos expression.

2. Results

2.1. Sxl-Pe is ectopically activated in the soma of nos

mutant 1X/2A embryos

In wild type, newly formed pole cells shut down RNA

polymerase II and remain transcriptionally inactive until

midway through embryogenesis (Seydoux and Dunn, 1997;

Van Doren et al., 1998). Maternally derived Nos is required

to establish/maintain this transcriptional quiescence. In

embryos from nosK mothers genes like even-skipped (eve)

and fushi tarzu (ftz) that are normally active only in the soma

are inappropriately expressed in pole cells at the syncytial

blastoderm stage. Though pole cells have the highest

concentration of Nos, there is a Nos protein gradient

extending to near the middle of the syncytial blastoderm

embryo. Consequently, the inhibitory effects of Nos in the

germline suggest that it may also suppresses transcription in

the soma.

Consistent with this idea we found that both eve and ftz

are inappropriately expressed in the posterior soma of

nosmK blastoderm embryos (not shown). However, since

translation of maternal hb mRNA drastically alters seg-

mentation in nosmK embryos, the activation of eve and ftz

could be due to excess Hb protein rather than to a failure in

downregulating transcription. Since hb does not have a

positive role in Sxl-Pe regulation (not shown) we examined

Sxl expression in the soma of nosmK embryos. In wild-type

blastoderm embryos Sxl expression is driven from the Sxl-

Pe promoter. As illustrated in Fig. 1, it directs the

production of Sxl protein in female but not male embryos.

Whereas Sxl is only observed in 50% of wild-type embryos,

it is found in the soma of all blastoderm stage nosm embryos.

This finding indicates that Sxl must be expressed in both

female and male nosmK embryos.

The nosm embryos could nevertheless be divided into two

equal classes based on protein levels. Embryos in the first

class have high levels of Sxl distributed uniformly

throughout the soma (Fig. 1). As embryos in this

class appear to have even more Sxl than wild-type

females, a reasonable presumption is that they correspond

to nosmK females. Embryos in the second class have lower

levels of Sxl than wild-type females (see Fig. 1) and thus are

likely to correspond to nosmK males. The ‘male’ embryo

shown in Fig. 1 has a relatively sharp posterior to anterior

gradient; however, the gradient in other male embryos can

be quite shallow and extend to the very anterior. Never-

theless, even these embryos can be readily distinguished

from wild-type males which have no detectable Sxl, and

from either nosmK ‘females’ or wild-type females which

have considerably more Sxl.

Fig. 1. Sxl protein is ectopically expressed from Sxl-Pe in the soma and pole cells of nosmK embryos. Panels in the top half show syncytial blastoderm embryos

stained with Sxl antibody. 0–4 h old embryos laid by white1 (w1) and nosBN mothers were probed with a monoclonal antibody directed against Sxl and the

staining pattern visualized by standard DAB immunohistochemical detection. As described in the text, wild-type embryos can be divided into two equal classes

based on the Sxl antibody-staining pattern in the soma. Embryos in the first class are darkly stained and previous studies demonstrate that these correspond to

females. Embryos in the second class are unstained. These are male embryos. Embryos from nosBN mothers can also be divided into two equal classes. Embryos

in the first class are darkly stained. As these embryos represent 50% of the population and resemble wild-type female embryos, we presume that they

correspond to nosBN female embryos (see also Fig. 2). Embryos in the second class are lightly stained (and sometimes exhibit a posterior to anterior gradient).

As these embryos also represent 50% of the population and resemble neither wild-type females, nor wild-type males, we believe that they correspond to nosBN

male embryos. To rule out the possibility that the low level of Sxl protein seen in these nosmK ‘male’ embryos is due to background staining, we mixed wild

type and nosmK before staining. As expected two classes of male embryos were observed in the mixture. The first class resembled wild-type males and had no

detectable Sxl. The second class resembled nosmK male embryos and had a low level of Sxl, often distributed in a shallow gradient. Panels in top half: WT

female: w1 female embryo showing a high level of Sxl protein in somatic nuclei and no protein in pole cells. WT male: w1 male embryo lacks Sxl protein. nos

‘female’: nosmK female embryo has Sxl in soma and pole cells. nosK male: nosmK male embryo has a posterior to anterior gradient of Sxl. Bottom panels show

blastoderm embryos stained with b-galactosidase antibody. nosBN or w1 virgin females were mated with males homozygous for Sxl-Pe3.0 kbLac-Z (Keyes et al.,

1992). 0–4 h old embryos were collected and stained with b-galactosidase antibody. WT male: no b-galactosidase is observed in either the soma or pole cells.

The nosmK embryos fell into two classes. The first class resembles wild-type females, and has high levels of b-galactosidase in the soma. However, unlike wild

type, b-galactosidase is also observed in the pole cell of these embryos. Based on their similarity to wild-type females and the fact that w50% of the nosmK

embryos fall into this class, we believe that these embryos are female. Embryos in the second class differ from wild type of either sex in that there is a low level

of b-galactosidase in both germline and soma. As observed for Sxl, there is often a posterior gradient. Since these embryos also represent 50% of the population

they are likely to correspond to nosmK males. Two nosmK male embryos are shown.

G. Deshpande et al. / Mechanisms of Development 122 (2005) 645–657 647

Since Nos is known to repress translation, the Sxl protein

in male progeny of nosK mothers could arise from the

translation of maternally derived Sxl mRNA rather than

from the activation of Sxl-Pe. To demonstrate that Sxl-Pe is

inappropriately turned on, we crossed nosBN females with

males carrying the full length Sxl-Pe3.0 kbLac-Z reporter.

In wild-type embryos, the Sxl-Pe reporter mimics

the endogenous Sxl-Pe promoter, driving b-galactosidase

expression in 2X/2A but not 1X/2A animals (Estes et al.,

l995; Table 1A; see also WT male in Fig. 1). In contrast,

since b-galactosidase can be detected in nearly 100% of

the nosmK embryos (Table 1A), Sxl-Pe3.0 kb must be

activated in both sexes. As is the case for Sxl protein, two

different b-galactosidase expression patterns are observed.

Table 1A

Sxl-Pe3.0 kb

Staining

Maternal genotype Dark Med.–

light

Negative Total

nosBN 64 (57%) 43 (38%) 5 (4%) 112

w1 156 (45%) 194 (55%) 350

FRT hbFBnosBN 47 (39%) 66 (56%) 8 (7%) 121

P-nos; FRT

hbFBnosBN

29 (47%) 7 (11%) 26 (42%) 62

nosBN, hbFBnosBN (females carrying germline clones for hbFBnosBN), P-nos;

FRT hbFBnosBN and w1 virgin females were mated with males carrying two

copies of Sxl-Pe3.0 kb transgene. Embryos derived from each cross were

stained for b-galactosidase activity. Based on the intensity of staining

embryos were classified into several categories.

Fig. 2. Expression of Sxl in wild type and nosK embryos. Wild type and

nosBN females were mated to wild-type males carrying an X-linked

ftz:hsp70LacZ transgene (Hagstrom et al., 1996). The UPS enhancer in this

transgene drives b-galactosidase expression in stripes in germ band

extending embryos. In this experiment, only female embryos carry the

ftz:hsp70LacZ transgene. Embryos collected from each cross were stained

with Sxl and b-galactosidase antibodies and examined by confocal

microscopy. Wild-type blastoderm stage embryos could be divided into

two equal classes based on Sxl antibody staining. Embryos in the first class

are female and express Sxl in the soma, but not the pole cells. Embryos in

the second class are male and Sxl cannot be detected. Unlike wild type, Sxl

can be detected in pole cells of all nosmK blastoderms. Based on the amount

of Sxl in the soma, the nosmK embryos can be divided into two equal

classes. Embryos in the first class express very high levels of Sxl protein

and as illustrated in Fig. 2 much greater amounts of Sxl accumulate in these

embryos than in wild-type females. These nosmK embryos are thought to be

female. Embryos in the second class also express Sxl; however, the amount

of Sxl is considerably less than wild type or nosmK ‘females’, and is often

most highly concentrated apically. These nosmK embryos are thought to be

males. The two panels at the bottom show germ band extended wild type

and nosmK embryos. At this stage all of the wild type and nosmK embryos

that had high levels of Sxl also expressed b-galactosidase and hence are

female. Note that as was observed at the blastoderm stage, nosmK female

embryos have much higher levels of Sxl than wild-type females. The

expected number (w50%) of male wild type and nosmK embryos lacking

b-galactosidase were also observed (not shown). While the wild-type males

had no Sxl, many of the nosmK male embryos had some residual Sxl.

G. Deshpande et al. / Mechanisms of Development 122 (2005) 645–657648

In slightly more than half of the blastoderm embryos high

levels of b-galactosidase are present throughout the soma

(see example in Fig. 1 and Table 1A). These nosmK embryos

resemble wild-type females. The remaining embryos have

lower levels of b-galactosidase (Fig. 1 and Table 1A).

To confirm these findings we examined Sxl expression

using confocal microscopy. In the experiment shown in

Fig. 2, males carrying an X-linked ftz(UPS):hsp70-LacZ

transgene (Hagstrom et al., 1996) were crossed to wild type

or nosBN females. The ftz UPS stripe enhancer activates the

hsp70 promoter and b-galactosidase expression can be

detected in gastrulating female embryos; however, it is not

observed prior to gastrulation. As illustrated in Fig. 2, Sxl

protein is present in the soma of wild-type female, but not

male embryos, while it cannot be detected in the pole cells

of either sex. Though Sxl in females localizes in the nucleus,

there are also substantial amounts in the apical and basal

cytoplasm at this stage. Unlike wild type, Sxl is present in

the soma and pole cells of all nosmK embryos. As found

with DAB staining, the nosmK blastoderm embryos could be

divided into two equal classes based on the level and pattern

of Sxl accumulation in the soma. Embryos in ‘Class I’ had

high levels of Sxl protein in the nucleus and surrounding

cytoplasm. Though the distribution of protein in these

embryos is similar to wild-type females, the amount of

Sxl, as judged by antibody staining, is higher (compare wild

type and nosmK female embryos in Fig. 2). Embryos in

‘Class II’ had much lower levels of Sxl than wild-type

females and much of the protein is localized in the apical

cytoplasm (Fig. 2).

We also examined later stages of development when

b-galactosidase expression from the ftz(UPS):hsp70-LacZ

transgene can be reproducibly detected. As illustrated at the

bottom of Fig. 2, for both wild type and nosmK, the germ

band extended embryos that expressed high levels of Sxl

were b-galactosidase positive. Consequently, these Sxl

expressing embryos must be female. Moreover, as was

observed at the blastoderm stage, the level of Sxl protein, as

judge by antibody staining, in older nosmK females is

consistently higher than in older wild-type females.

As expected, the male, b-galactosidase negative, germ

band extended embryos in the wild-type collection had no

Sxl. In the nosmK collection, residual Sxl protein could be

detected in some of the b-galactosidase negative males

Table 1B

Sxl-Pe0.4 kb

Maternal

genotype

Very

dark

Dark Medium Light Negative

nosBN 35 (20%) 35 (20%) 47 (28%) 49 (29%) 2

hbFBnosBN 23 (21%) 31 (29%) 23 (21%) 24 (21%) 5

w1 205 (45%) 0 0 259 (55%)

G. Deshpande et al. / Mechanisms of Development 122 (2005) 645–657 649

(not shown); however, the level of Sxl was considerably

lower than in the blastoderm ‘Class II’ nosmK embryos.

These findings indicate that the amount of Sxl expressed

from Sxl-Pe in nosmK male blastoderm embryos is generally

not sufficient to stably activate the autoregulatory feedback

loop.

2.2. Activation of the minimal promoter, Sxl-Pe0.4 kb

To further localize the nos responsive sequences we

tested a reporter containing the minimal Sxl-Pe promoter,

Sxl-Pe0.4 kb (Estes et al., 1995). While Sxl-Pe0.4 kb

exhibits the appropriate sex-specificity in wild type (see

Table 1B), it drives considerably less b-galactosidase

expression than Sxl-Pe3.0 kb and unlike the larger

promoter whose activity is nearly uniform, Sxl-Pe0.4 kb

is much more active in the anterior than the posterior. As

was the case for Sxl-Pe3.0 kb, Sxl-Pe0.4 kbLac-Z must be

inappropriately turned-on in males in the absence of Nos

function since it is active in nearly 100% of the nosmK

embryos (Fig. 3 and Table 1B). In addition, the level of

b-galactosidase in nosmK ‘female’ embryos is increased

relative to wild type. Notice that the effects of nos on

this promoter are not strictly limited to the posterior of

the embryo.

2.3. Ectopic expression of Nos protein downregulates

Sxl-Pe activity

If Sxl-Pe is upregulated in the absence of Nos, it should

be repressed by excess Nos. To test this prediction we used

nos transgenes in which the nos 3 0UTR is replaced by

a tubulin (tub) or a bicoid (bcd) 3 0UTR. In progeny from

nos-tub 3 0UTR mothers ectopic Nos protein is produced

throughout the embryo, while in progeny from nos-bcd

3 0UTR mothers ectopic Nos is translated from transgene

mRNA localized at the anterior.

We first examined the effects of excess Nos on Sxl-Pe

activity in 2X/2A embryos. Though about 50% of the

blastoderm embryos produced by nos-tub 3 0UTR or nos-bcd

3 0UTR mothers still express Sxl protein, most show striking

abnormalities in the pattern of protein accumulation. In the

case of nos-tub 3 0UTR, Sxl expression is often reduced in

the posterior (see arrows in Fig. 4). However, we also

observe embryos in which there is a general reduction in

Sxl protein throughout or show a patchy pattern of Sxl

protein accumulation several different regions (not shown).

In the case of nos-bcd 3 0UTR, female embryos in which Sxl

protein is reduced in the anterior are often observed (Fig. 4).

These findings argue that ectopic Nos downregulates Sxl-

Pe. Consistent with this idea, Nos from the nos-bcd 3 0UTR

(Fig. 3) or nos-tub 3 0UTR (not shown) transgenes represses

the Sxl-Pe3.0 kb reporter.

Since activation of the normally silent Sxl-Pe in 1X/2A

embryos is one of the most striking effects of reduced Nos,

we wished to determine whether excess Nos downregulates

Sxl-Pe in male embryos as it does in female embryos.

However, this experiment is not possible with the wild type

Sxl-Pe because it is already off in nosC 1X/2A embryos. For

this reason, we took advantage of a ‘gain-of-function’

promoter, Sxl-PeGOF, that has extra numerator binding sites

and is active in both sexes (Jinks et al., 2000; Kramer et al.,

1999). As shown in Fig. 3, Sxl-PeGOF is repressed in 1X/2A

embryos by excess Nos.

2.4. Phosphorylation of the RNA polymerase CTD domain

is altered in nos embryos

The upregulation of transcription in nosmK embryos

suggested that it would be of interest to examine ser2 and

ser5 phosphorylation of the CTD domain since these

modifications of the largest RNA polymerase II subunit

are correlated with transcriptionally engaged polymerases.

As reported by Seydoux and Dunn (1997), phospho-ser2 is

detected in somatic nuclei of wild-type blastoderm embryos,

but not in pole cell nuclei (Fig. 5). This is not due to a

germline specific reduction in the amount of polymerase II

as pole cell nuclei are labeled to the same extent as somatic

nuclei by an antibody that recognizes epitopes in exon 2 of

the large subunit (not shown). Unlike wild type, the

phospho-ser2 CTD modification is readily detected in

nosmK pole cells (Fig. 5, see legend). In stage 4 embryos

roughly 50% of the pole cells are stained have the CTD

phospho-ser2 modification while the number and level

increases in stage 5 embryos. We also probed for the

phospho-ser5 modification (not shown). As observed in the

C. elegans germline, only a low level of phospho-ser5 is

found in wild-type pole cells, while it is elevated in a subset

of nosmK pole cells.

The effects of nos on CTD phosphorylation are not

limited to the germline. The level of phospho-ser2 antibody

staining in somatic nuclei of syncytial blastoderm nosmK

embryos is upregulated compared to syncytial wild-type

embryos stained in parallel (Figs. 5 and 6). This increase is

readily detected in the posterior of the embryo where Nos

protein levels are maximum. Since nosmK and wild-type

embryos are labeled to the same extent by the exon2-

specific polymerase antibody (Fig. 7), the increase in

phospho-ser2 in the soma of nosmK embryos is not due to

differences in the amount of the large subunit. We also

found that phospho-ser5 appears to be increased in nosmK

(not shown).

Fig. 3. Top: The minimal Sxl-Pe0.4 kb promoter is upregulated in nosmK embryos. Homozygous nosBN and w1 females were separately mated with males

homozygous for Sxl-Pe0.4 kbLac-Z. 3–7 h old embryos from each cross were stained for b-galactosidase. In wild type, the Sxl-Pe0.4 kbLac-Z reporter drives b-

galactosidase expression in female, but not in male embryos. A different result is obtained in embryos from nosmK mothers; b-galactosidase is expressed in

female and male embryos. In addition, the level of b-galactosidase in female embryos from nosmK mothers is higher than in female embryos from wild-type

mothers. Middle: Ectopic Nos can downregulate Sxl-Pe3.0 kb in female embryos. w1 (WT) and nos-bcd 3 0UTR females were mated with males homozygous for

Sxl-Pe3.0 kb:Lac-Z. 3–7 h old embryos from each cross were stained for b-galactosidase. In w1 (WT) embryos, the Sxl-Pe Lac-Z reporter drives b-galactosidase

expression in female, but not in male embryos. A different result is obtained in progeny of nos-bcd 3 0UTR mothers; b-galactosidase is reduced and patchy in

female embryos. Bottom: Ectopic expression of Nos protein can downregulate a ‘gain of function’ form of Sxl promoter in male embryos. w1 (WT) and nos-bcd

3 0UTR females were mated with males homozygous for the ‘gain of function’ Sxl-PeGOF:Lac-Z reporter. In w1 (WT) embryos, Sxl-PeGOF drives characteristic

patterns of b-galactosidase expression in female (not shown), and male embryos (see Figure). As illustrated by the nos-bcd 3 0UTR embryo on the right, ectopic

Nos reduces b-galactosidase expression from Sxl-PeGOF in male embryos.

G. Deshpande et al. / Mechanisms of Development 122 (2005) 645–657650

To confirm these results, we compared the relative amount

of CTD phospho-ser2 in 0–3 h nosmK and wild-type embryos

by Western blotting using as an internal control either Snf

(Fig. 6) or yolk protein (not shown). We found that the level

of phospho-ser2 is about 1.5-fold higher in nosmK embryos

than in wild type. Although the increase in CTD phosphoryl-

ation measured by Western is small, differences between

nosmK and wild type were consistently observed.

2.5. CTD phosphorylation is downregulated by ectopic Nos

Because the changes in CTD phosphorylation in the

soma of nosmK embryos are modest, we sought additional

evidence that Nos regulates this modification. If the

absence of Nos is correlated with increased CTD

phosphorylation, we reasoned that phosphorylation

should be reduced when Nos is ectopically expressed.

Fig. 4. Ectopic expression of Nos protein can downregulate Sxl expression

in somatic nuclei of female embryos. Panels on left: 0–4 h old embryos

collected from w1 wild type and nos-tub 3 0UTR females were stained with

Sxl antibody. In WT female embryos all somatic nuclei express Sxl;

however Sxl is absent in pole cells (top left photo). The effects of ectopic

Nos vary somewhat from embryo to embryo. In the example shown here

(bottom left photo) the nos-tub 3 0UTR embryo shows a reduction in Sxl

protein in the posterior as indicated by the arrows. Panels on right: 0–4 h old

embryos collected from w1 wild type and nos-bcd 3 0UTR females were

stained with Sxl antibody. In WT female embryos all somatic nuclei

express Sxl, while Sxl is absent in pole cells (top right photo). In the nos-

bcd 3 0UTR embryo (bottom right photo) Sxl protein is reduced in the

anterior as indicated by the arrows. Note the effects of Nos expressed from

the nos-bcd30UTR are not always restricted to the anterior and can extend

through the entire embryo.

G. Deshpande et al. / Mechanisms of Development 122 (2005) 645–657 651

This prediction is correct. For the nos-tub3 0UTR trans-

gene, the phospho-ser2 antibody-staining pattern is

typically irregular and patchy; some regions of the

embryo have near wild-type levels of phosphorylation,

while others do not. Moreover, this reduction in

Fig. 5. CTD phosphorylation in wild type and nosmK embryos. 0–4 h

embryos laid by w1 and nosBN mothers were probed with a monoclonal

recognizing phospho-ser2 of the CTD repeats (H5) and polyclonal Vasa

antibody. Left panels: Vasa is imaged in green while phospho-ser2 is

imaged in red. WT embryo stained with H5 antibody showing staining in

the somatic nuclei. However, there is little phospho-ser2 in the pole cells.

By contrast, in nosmK embryos phospho-ser2 is observed in both somatic

and pole cell nuclei. In stage 4 embryos roughly 50% of the total number of

nosmK pole cells are labeled with the anti-phospho-ser2 antibodies. Right

panels: Surface views of phospho-ser2 in the somatic nuclei of wild type

and a nosBN embryos. The embryos were stained and imaged in parallel

under the same conditions.

phospho-ser2 is observed even in the absence of endogenous

Nos protein (see arrows in nosK; nos-tub3 0UTR embryos in

Fig. 7). For the nos-bcd 3 0UTR transgene, a subset of

embryos phospho-ser2 is diminished in the anterior (Fig. 8).

Most of the remaining embryos showed a more general

reduction in phospho-ser2l compared to wild type. Ectopic

Nos also causes a reduction in the level of phospho-ser5. As

shown in Figs. 7 and 8, ectopic Nos expression from the nos-

bcd 3 0UTR transgene reduces phospho-ser5 at the anterior.

Since exon2 antibody staining is unaltered by ectopic Nos

(Fig. 7), the alterations in phospho-ser2 and phospho-ser5

seen in nos-tub3 0UTR and nos-bcd3 0UTR embryos are not

due to a block in the translation of the large polymerase

subunit mRNA.

2.6. Does transcriptional activation in the soma

disrupt development?

The results in Section 2.5, together with similar findings

for eve and ftz, suggest that the activity of the transcriptional

apparatus is elevated in the absence of nos. Though the early

embryo is known to have compensatory mechanisms to deal

with excesses or shortfalls in key gene products, it seemed

possible that an increase in expression of many patterning

genes might have detrimental consequences. Indeed,

previous studies by Irish et al. (1989) have shown that

while defects in the posterior development of nosmK

embryos can be suppressed by eliminating maternal hb,

viability is not completely restored and only about 45% of

the embryos carrying a wild-type paternal hb gene reach the

adult stage. However, Irish et al. (1989) generated the nosK

hbK female germ line by pole cell transplantation and this

incomplete rescue could be due to abnormalities in the eggs

produced by germ cells damaged during transplantation. For

this reason, we reexamined this question using FRT to

generate germline clones.

In the first experiment we generated germline clones

for nosK hbK, and as a control hbK alone. The females

were then mated to wild-type males. Less that 60% of the

progeny of nosK hbK germline clone mothers hatched as

1st instar larva (see Table 2). By contrast, about 85% of

the progeny of hbK mothers hatched. If the reduced

viability of embryos from nosK hbK mothers is due to the

lack of nos activity, it should be possible to increase their

viability by providing nos in the mother with a nos rescue

transgene. As can be seen in Table 2, this is the case. In

the presence of the nos transgene the percentage of

progeny from nosK hbK germline clone mothers that

hatch is increased to about 90%. We also compared Sxl-

Pe activity in the progeny of nosK hbK mothers with and

without the nos rescue transgene. As observed in nosK

embryos, we found that Sxl-Pe is active in both sexes in

the progeny of nosK hbK germline clone mothers (Tables

1A and B). In contrast, embryos produced by nosK hbK

mothers carrying the nos rescue transgene resemble wild

type in that Sxl-Pe is active in only 50% of the embryos.

Fig. 6. Phosphorylation of serine 2 in CTD repeats of RNA Polymerase II in wild type and nosmK mutant embryos. Panel on left: 0–4 h embryos from w1 and

nosKBN mothers were stained with monoclonal (H5) specific for CTD repeats that are phosphorylated on serine 2. Top: WT embryo probed with the H5

monoclonal. Wild-type embryos show near-uniform staining of somatic nuclei and little or no staining of pole cell nuclei. The two embryos labeled nos are

nosmK embryos probed with H5 antibody. Note that the staining appears to be stronger in the somatic nuclei of nosmK embryos than in the wild-type control.

(H5 staining of somatic nuclei in wild type and nosmK embryos sometimes appears stronger on the dorsal side.) Panels on right: Embryonic extracts prepared

from 0 to 2.5 h old embryos were resolved using 7% SDS polyacrylamide gel electrophoresis, blotted onto nitrocellulose and probed with H5 antibody. After

visualization of the H5 signal, the same filter was probed with Snf antibody. Left panel: Extracts from embryos laid by WT mothers. Right panel: nosmK

embryo extracts. These embryos were laid by nosBN mothers. Individual band intensities were measured and averaged using NIH image analysis. (119.89G

31.60 for the control samples and 187.20G33.06 for the experimental samples). The normalization was performed using corresponding band intensity values

obtained using Snf as a control. The normalized enrichment was estimated to be 1.48-fold.

G. Deshpande et al. / Mechanisms of Development 122 (2005) 645–657652

In a third experiment we followed embryos produced from

mating nosK hbK germline clone mothers to wild-type

males from hatching to the adult stage. In this experiment,

only about 55% of the 457 progeny hatch as first instar

larva. Of the unhatched embryos, approximately 25% had

knirps-like posterior defects typical of that produced by

Hb misexpression (see Fig. 9). A range of defects was

observed in the remaining embryos. Some had near wild-

type cuticles, while others formed only scraps of cuticle

or exhibited a variety of segmentation defects such as

fusions or deletions of multiple segments. Only about

42% of the progeny survived to the adult stage. As might

be expected from the Sxl expression pattern at mid-

embryogenesis, activation of Sxl-Pe in male nosK

blastoderm embryos had no lasting consequences, and

ratio of male to female adults was almost 50/50.

3. Discussion

During the rapid nuclear division cycles in cleavage

stage Drosophila embryos RNA polymerase II transcription

is largely shut down and only a few genes are actively

transcribed. RNA polymerase II transcription in somatic

nuclei is upregulated soon after they migrate to

the periphery of the embryo at stage 9 and by nuclear

cycle 10 and 11 many of the key segmentation genes are

already actively transcribed (Lamb and Laird, l976;

Zalokar, l976). While RNA polymerase II activity is

substantially augmented when the nuclei reach the periph-

ery of the embryo in the soma, the opposite occurs in the

germline pole cell nuclei. When these nuclei migrate into

the posterior pole plasm and pole cells are formed,

transcription is shut down rather than activated (Williamson

Fig. 7. Ectopic Nos downregulates Ser2 and Ser5 CTD phosphorylation. Ser2 CTD Phosphorylation: 0–4 h old embryos from w1 and nosBN; nos-tub 3 0UTR

mothers were stained with a monoclonal (H5) recognizing phosphorylated serine at position 2 in the CTD repeats of the large RNA polymerase II subunit. The

H5 antibody-staining pattern in a wild-type blastoderm embryo is in the top panel, while the two lower panels have embryos from nosBN; nos-tub 3 0UTR

mothers processed in parallel. Note that staining is reduced and patchy in the embryos from transgene mothers (see arrows). Ser5 CTD Phosphorylation: 0–4 h

old stage embryos laid by w1 and nos-bcd 3 0UTR mothers were stained with a monoclonal antibody (H14) recognizing phosphorylated serine at position 5 in the

CTD repeats of the large RNA polymerase II subunit. The H14 antibody-staining pattern in a wild-type blastoderm embryo is in the top panel, while the two

lower panels have embryos from nos-bcd 3 0UTR mothers processed in parallel. Note that staining is reduced and/or patchy in the anterior of embryos from

transgene mothers (see arrows). Note also that unlike H5, the H14 antibody labels wild-type pole cells, indicating that there is a low level of ser5

phosphorylation in wild-type germ cells. Exon 2: The polyclonal ‘ex2’ antibody recognizes epitopes encoded by exon 2 of the large RNA polymerase II

subunit. Shown on the far right is ex2 antibody staining of embryos from wild type, nosBN and nos-tub3 0UTR mothers. Note that the pattern and intensity of

staining in embryos from both nosBN and transgene mothers is indistinguishable from wild type. Though not readily visible at the magnification shown in these

photographs, the pole cells of all embryos are labeled by the ex2 antibody.

G. Deshpande et al. / Mechanisms of Development 122 (2005) 645–657 653

and Lehmann, 1996). Previous studies have implicated the

posterior determinants nos and pum in establishing/main-

taining transcriptional quiescence in pole cells (Deshpande

et al., 1999; Asaoka et al., 1998, 1999). In embryos derived

from mothers mutant for either nos or pum, RNA

polymerase II transcription is not properly downregulated

in the pole cells and several genes which are normally active

only in somatic nuclei are ectopically expressed (Parisi and

Lin, 2000).

Since only nos mRNA localized at the posterior pole is

translated, pole cells have the highest levels of Nos.

However, translation of the localized message generates a

Nos gradient that extends to the center of the embryo. An

obvious question is whether this Nos gradient also affects

RNA polymerase II activity in somatic nuclei. Indeed,

transcription of Sxl-Pe is upregulated in somatic nuclei

when Nos protein is removed, and is repressed when Nos

protein is ectopically expressed. The role of Nos protein

in repressing transcription is not restricted to the sex

determination pathway since the activity of other

promoters also appears to be increased in the absence of

nos function.

3.1. Mechanism of activation

Several mechanisms could potentially explain the

ectopic activation of Sxl-Pe in the soma and germline of

nos mutant embryos. The most obvious is that this

promoter is turned-on by maternal Hb expressed in the

absence of nos. However, Sxl-Pe was upregulated in nosK

embryos even when we eliminated maternal Hb. In

addition, ectopic expression of Hb from a transgene

lacking NREs seemed to repress rather than activate Sxl-

Pe (not shown). Another possibility is that the zygotic

expression of one or more of the X-linked numerators is

elevated in nos embryos, upsetting X chromosome to

autosome counting. However, as none of the known

numerators has a recognizable NRE in the 3 0UTR of its

message, it seems unlikely that these genes are direct

targets for translational repression by Nos protein.

In addition, it is not at all clear why numerator genes

(which are transcribed in the zygote) would be subject

to translational repression by Nos, while autosomal

denominator genes such as deadpan (which turns off

Sxl-Pe) would not.

Fig. 8. Downregulation of Ser2 and Ser5 CTD Phosphorylation in the anterior in embryos from nos-bcd 3 0UTR mothers. Top panels: confocal images of

anterior and posterior halves of a blastoderm stage embryo from nos-bcd 3 0UTR mother probed with antibodies against CTD repeats containing the phospho-

ser2 modification. Bottom panels: confocal images of the anterior half of a blastoderm embryo from nos-bcd 3 0UTR mothers stained with the DNA dye Hoechst

(blue) and antibodies against CTD repeats containing the phospho-ser5 modification (green).

Table 2

Germline clone analysis

Total #Hatched % Hatched

Experiment I

FRT hbFBnosBN!w1 1079 625 58

FRT hbFB!w1 1580 1339 85

Experiment II

FRT hbFBnosBN!w1 69 39 57

P-nos; FRT hbFBnosBN!w1 421 388 92

G. Deshpande et al. / Mechanisms of Development 122 (2005) 645–657654

For this reason, we favor the idea that Sxl-Pe is activated

in nosmK embryos at least in part because RNA polymerase

II activity is upregulated. Support for this idea comes from

analysis of CTD phosphorylation. When RNA polymerase

is transcriptionally engaged the CTD domain is phosphory-

lated on serine 2 and 5. In wild-type pole cells, phospho-ser2

cannot be detected, while there is only little phospho-ser5.

In contrast, phospho-ser2 is found in nosmK pole cells, while

the level of phospho-ser5 is increased. nos-dependent

alterations in CTD phosphorylation are also evident in the

soma. When Nos is absent, the level of ser2 and ser5 CTD

phosphorylation is elevated, while both types of CTD

phosphorylation are reduced by ectopic Nos protein.

Additional evidence that nos has a global effect on

transcription comes from the finding that nos regulates the

methylation of histone H3 in the germline of worms and

flies (Schaner et al., 2003). In both organisms, the

methylation of histone H3 on lysine 4 (H3meK4) is

upregulated in the soma when zygotic transcription

commences in early embryogenesis. In contrast, little or

no methylation H3 K4 is observed in the transcriptionally

quiescent germline. Inhibition of H3 K4 methylation in

germ cells requires nos and H3meK4 is markedly

upregulated in nosK germ cells. In light these findings, we

examined K4 methylation in the soma of nosmK embryos.

Fig. 9. Cuticular defects observed in embryos derived from females

carrying germline clones for hbFBnosBN. Females carrying germline clones

for hbFBnosBN were mated with wild-type males. Embryos derived from

this cross were allowed to develop (see Table 2). The photos show four

different examples of cuticles. (A) Embryo displaying a near normal

segmentation pattern. (B) Embryo with fusions resembling a weak ‘knirps-

like’ phenotype. (C) Embryo with more severe segmentation defects.

(D) Embryo that lacks several segments.

G. Deshpande et al. / Mechanisms of Development 122 (2005) 645–657 655

As might be expected from the effects of nos on CTD

phosphorylation, somatic H3meK4 is elevated compared to

wild type (Desphande, unpublished data).

Since phosphorylation of serines 2 and 5 are correlated

with transcription, the nos-dependent alterations in CTD

phosphorylation are consistent with the idea that nos has

a global impact on RNA polymerase II activity. If this is

the case, an important question is whether CTD

phosphorylation is the cause or the consequence of nos

induced changes in the activity of the transcriptional

apparatus. Because actively transcribing RNA polymerase

has a hyperphosphorylated CTD domain, any mechanism,

which leads to a general increase (or decrease) in

transcription, would likely alter the level of CTD

phosphorylation. This makes it difficult to distinguish

between cause and effect. On the other hand, besides

being a characteristic feature of elongating polymerase,

CTD phosphorylation has been linked to the last steps in

the initiation process, promoter clearance and the

formation of an elongation competent RNA polymerase

complex. Moreover, there is growing evidence that these

steps in the transcription cycle are subject to regulation

(Lee and Lis, l998; Batchelder et al., 1999; Nissen and

Yamamoto, 2000; Shim et al., 2002).

The fact that CTD phosphorylation may be a key

control point in the transcriptional cycle raises the

possibility that nos exerts its effects on polymerase

activity by inhibiting the translation of some factor

which promotes CTD phosphorylation. In nos mutants,

the level of this factor would increase, leading to a

general derepression of transcription. Conversely, the

level of this factor would decrease by ectopic Nos,

reducing overall transcription.

3.2. The Nos gradient and activation of Sxl-Pe

in nosmK embryos

While our results clearly show that Sxl-Pe is inappropri-

ately turned on in the soma of male embryos and

upregulated in the soma of female embryos in the absence

of nos activity, it was initially surprising to find that there is

usually not a very pronounced posterior–anterior activation

gradient. In fact, the smaller Sxl-Pe0.4 kb promoter is clearly

activated not only in the posterior but also in the anterior of

nos embryos, while the larger Sxl-Pe3.0, usually shows at

most only a very shallow posterior–anterior gradient of b-

galactosidase expression. Since the Nos gradient does not

extend beyond the midpoint of the embryo, and the

repressive effects of Nos on hb mRNA translation are

restricted to this posterior domain, one might have expected

that the activation of Sxl-Pe in would be tightly restricted to

the posterior half of nos mutant embryos. However, proteins

of average size would be expected to diffuse (in water or

even in cytoplasm) through the volume of a fly embryo over

a time scale of minutes, and the establishment of gradients

like those seen for Nos or Bcd are likely to require special

mechanisms including a localize source of product, as well

as the sequestration (e.g. nuclear localization) and degra-

dation of the product. If the Nos target for inhibiting general

RNA Pol II activity is translated from a uniformly

distributed maternal mRNA and is able to equilibrate

through the embryo during the time between the onset of the

very rapid nuclear divisions and the formation of the cellular

blastoderm, only a shallow gradient of this factor in the

soma might be expected at any one time in the presence or

absence of Nos. In contrast, in pole cells, where repression

of this factor by Nos would presumably be required to

impose transcriptional quiescence, the formation of the cell

membrane would prevent factor synthesized in the soma

from influencing polymerase activity. This would enable

Nos in the pole cells to reduce the level of this factor below

the threshold required for transcriptional activation.

3.3. Transcriptional repression in the soma

As observed in many species, establishing transcriptional

quiescence in the newly formed pole cells during early

embryogenesis is a critical step in the development of the

Drosophila germline. However, it is not immediately

obvious what role nos mediated down regulation of

polymerase activity would have in the development of the

soma. Obviously, hyperactivation of Sxl-Pe in nosmK

embryos could inappropriately switch on the Sxl autoregu-

latory feedback loop in males. However, our analysis of Sxl

accumulation in post-blastoderm stages suggests that only

very few nosmK 1X/2A embryos actually make the wrong

choice in sexual identity. There is also little evidence of a

sex-bias in adult progeny of nosK hbK germline clone

mothers. The fact that the Sxl autoregulatory loop is usually

not activated in male nosmK embryos, which are

G. Deshpande et al. / Mechanisms of Development 122 (2005) 645–657656

hemizygous for Sxl, is not altogether surprising. In females

that have only a single wild type Sxl gene, activation of the

autoregulatory loop is severely compromised by conditions

which diminish Sxl-Pe activity (Cline, 1988). Since the

amount of Sxl produced by Sxl-Pe in nosmK male embryos

is much less than that in wild-type females, the auto-

regulatory loop should be activated infrequently.

While nosmK males largely escape the effects of

activating Sxl-Pe, the increased polymerase activity appears

to have other consequences. Previous studies have shown

that removal of maternal hb suppresses the posterior defects

of nosmK embryos (Hulskamp et al., 1989; Irish et al., 1989;

Struhl, 1989). However, in the experiments of Irish et al.

(1989) only about 40% of the hbC/hbK embryos from nosK

hbK mothers survive to adults. Likewise, we have found

that only 60% of the embryos produced by hbK nosK

mothers hatch as first instar larva, and that an even lower

number survive to the adult stage. Taken together, these

experiments argue that nos has important functions in the

soma besides blocking translation of maternal hb mRNA. It

seems possible that the segmentation/developmental defects

evident in progeny of hbK nosK clone mothers could arise

from the upregulation of various patterning genes in the

absence of nos activity. We presume that in wild-type

embryos the activity of the transcriptional apparatus and of

target zygote promoters is appropriately adjusted to

compensate for the repressive effects exerted by Nos.

Because the transcriptional apparatus is hyperactivated in

the absence of Nos function, this balance is perturbed and

many genes are overexpressed.

4. Experimental procedures

4.1. Strains and culturing

Flies were grown on a standard medium at 25 8C unless

otherwise noted.

4.2. Germline clonal analysis

Females carrying hbFBnosBN or hbFBnosBN; nosC

transgene germline clones were generated essentially as

described previously (Forbes and Lehmann, 1998).

4.3. Immunohistochemistry

Embryos were probed with antibodies essentially as

described in Deshpande et al. (1995). In order to confirm

any differences seen in the staining patterns between

embryos with different maternal nos genetic backgrounds

when they were stained independently, we mixed the

experimental and control samples together and then stained

the combined samples. In all cases, we observed the

expected mixture of ‘wild type’ and ‘mutant’ staining

phenotypes. Figures only show embryos stained

independently. Anti-Sxl antibody (Bopp et al., 1991) is a

mouse monoclonal and was used at 1:10 dilution. H5 and

H14, the antibodies against phosphorylated CTD repeats of

the large RNA polymerase II subunit were purchased from

Research Diagnostics Inc. Both the antibodies were pre-

absorbed against WT embryonic samples and were sub-

sequently used at 1:250 dilution. Anti-exon 2, a goat

polyclonal antibody (1:100) against the large subunit of

PolII, was a generous gift of Dr Arno Greenleaf.

4.4. Western blot analysis

The Western blot analysis was performed essentially as

described in Deshpande et al. (1995). Embryonic extracts,

prepared from 0 to 2.5 h old embryos, were resolved using

7% SDS polyacrylamide gel electrophoresis, blotted onto

nitrocellulose and probed with H5. Typically, total embryo-

nic extract made out of 2 embryos was loaded per lane. To

compare the total protein loaded onto each lane, the blots

were subsequently reprobed with Snf antibody (1:10). The

intensity of individual bands was measured using NIH

image program and the ratios were calculated to estimate the

fold increase.

Acknowledgements

We thank Gary Struhl, Paul Macdonald, Paul Lasko and

Arno Greenleaf for antibodies, Claude Desplan, Liz Gavis,

Judith Lengyel, and Robin Wharton for fly stocks. We

would like to thank Trudi Schupbach, Eric Wieschaus, Liz

Gavis and Daryl Gohl for advice. We also thank Radhika

Mohan for help in preparing the manuscript. This work was

supported by a grant from the N.I.H. (P.S.).

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