[ LSF~ ' IFR St I [ N ( I IR I I ~ N [ ) Mechamsms of Development 46 (1994) 15-25

Genetic factors controlling the expression of the abdominal-A gene of Drosophila within its domain

Ana Macias*, Soraya Pelaz, Gin6s Morata

Centro de Blologta Molecular. Um~er~tdad Aut(moma de MadrM-CSIC. Madrid. Spurn

(Received 21 October 1993 rewslon recewed 27 November 1993. accepted 7 December 1993)

Abstract

The homeotlc gene abdommaI-A (abd-A) Is normally expressed m parasegments 7 to 13 We find that the lmtlal distribution of the product is approximately uniform within this domain, but the subsequent elaboration of the expression pattern results m differ- ences between, as well as w~thln, parasegments We have mvestlgated the possible role of several pa~r-rule, e g /usht tarazu, even- skipped, runt, hairy, paired, and segment polarity e g engratled, wmgles.s, naked, patched and cubttu.s mterruptus genes on the pattern- mg of abd-A expression We find that the estabhshment of the original ahd-A expression domain is independent of any of these genes, but most of them are required for the subsequent elaboration of abd-A expression w~thln the domain The genes [usht tarazu, and especially engratled, appear to act as transcriptional activating factors of abd-A

Key words Homeotlc genes, abd-A expression, Segment polarity genes, Pair-rule genes

1. Introduction

The activation of homeotic genes in certain body areas and their repression in others defines the genes characteristic expression domains (see Akam, 1987 and Lawrence, 1992 for general descriptions). The establish- ment of the expression pattern is a complex regulatory phenomenon involving several levels of control.

First, early acting maternal, gap and pair-rule genes probably recogmze specific sequences and render the homeotic gene accessible for transcription. For example, the early expression pattern of Deformed (Did) requires a combination of bicoid, hunchback (hb) and several pair-rule genes (Jack and McGxnnls, 1990), or the genes hb and fushl tarazu OCtz) play an important role in the establishment of early Ubx activity (Ingham and Martlnez-Arias, 1986, White and Lehman, 1986; Mfiller and Blenz, 1992). This first step is probably responsible of the definition of the expression domain and the establishment of the initial levels of expression

* Corresponding author

Second, genes functioning permanently during embryomc and larval development, such as the segment polarity and the homeotlc genes themselves, also con- tribute to the control of homeotic gene expression: the gene engraded(en), for example, is known to affect Ubv expression (Martinez-Arlas and White, 1988, Qlan et al., 1993). Among the homeotlc genes there are several cases of cross Interactions (Hafen et al., 1984, Struhl and White, 1985, Macias at al., 1990) resulting in down- or up-regulation of homeotlc genes transcription. This includes the ability of some homeotlc genes to auto- regulate their own expression (Kuz~ora and McGlnms, 1988; Bienz and Tremml, 1988; Choumard and Kauf- man, 1991, Irvlne et a l , 1993) This second step is prob- ably involved in the modulation during development of the homeotlc gene activity A tMrd level of control ~s represented by the PoO'comb (Pc) and Pc-group of genes, which are essentially repressors of homeotlc activity (Jurgens, 1985: Paro and Hogness, 1991)

The abdominal-A (abd-A) gene is a member of the bi- thorax complex (BX-C) involved in the specification of the abdominal region (Karch et al., 1985, Sanchez-

0925-4773/94/$07 00 © 1994 Elsevier Soence Ireland Ltd All rights reserved SSDI 0925-4773(94)00227-E

16 ,4 Mantas el al , Mechantsms o /Deve lopmen t 4 6 : 1 9 9 4 ) 15-25

Herrero et al, 1985, Busturla et al., 1989) It appears to code for only one protein form (Cumberledge et al, 1990), and its expression extends from parasegment 7 to 13 (Macias et al., 1990; Karch et al , 1990) The expres- ston of abd-A Is relatively homogeneous m &fferent parasegments of the domain, although parasegments 7 and 13 show less product than the rest Wlthm each parasegment there is a modulatton of the amount of product (Macias et al, 1990, Karch et al, 1990) whtch

gives an abd-A expressmn pattern retterated m each parasegment

In this paper we examine some aspects o1" the regula- tion ofabd-A expression, especially the factors respons> ble for the local differences of expression arising w~thm the domain Our results suggest that the segment pol,m- ty genes engratled (en) and wm~,,le~s (w~) have a role m the generation of the mtrametamerlc pattern ol ahd-,t expression, and that the parr-rule genes /l- and even-

F E C

D

Fig 1 ah~LA prote in (A and F) and R N A express ion ( B - E ) m different stages and germ layers (A) ahd-A prote in express ion ,it the ex tended germ

b a n d per iod An te r io r bo rde r s o f p a r a s e g m e n t s 7 and 13 are mdma ted by a r rows Not ice the g r admnt m the a m o u n t o f an t igen , f rom the h~gh level

m the a n t e r m r pa ra segmen ta l b o r d e r to the lowest level jus t a n t e r m r to the next p a r a s e g m e n t The a rea o f ahd-4 expressmn m p a r a s e g m e n t I~

is smaller than in the o thers ( B - D ) R N A express ion m stages 6, 7 a n d 10 It is app rox ima te ly unlfk~rm m stage 6 but m o d u l a u o n Lan alread5

be observed in 7 and ~t is well es tabl i shed by s tage I0 Not ice in D tha t the a m o u n t ot R N A m 13 is less than in the o ther pa r a segmcn t s , m parallel ,,vlth wha t Is observed at the pro te in level (F a n d E) C o m p a r i s o n o f the pro te in and R N A express ion In the ma tu re nerxc Lord The prote in cxpre,.-

slon s tops ab rup t ly In p a r a s e g m e n t 1t whereas low levels o f R N A can still be observed in pa r a segmen t s 14 and 15

A Maciaa et al /Me~ham~nl~ o! Development 46 (1994) 15-25 17

skipped (eve) are required for establishing the level of abd-A product in different parasegments.

2. Results

2 1 Wtldtype expresston of abd-A

The normal &strlbution of abd-A protein has already been described using a specific anti-abd-A antibody (Maclas et a l , 1990; Karch et al., 1990), and is illus- trated m Fig. 1 The anterior limit coincides exactly with parasegment 7, whereas the posterior limit runs throughout the anterior compartment of parasegment 13 (A8a), but does not coincide with any segmental or parasegmental boundary. There are also differences be- tween metameres, for example, there is less protein in parasegment 7 than m parasegments 8-12, and in 13 the expression In A7p is high, but in A8a only some few cells contain detectable amount of abd-A product (Macias et al. 1990) Within each metamere (except m parasegment 13) abd-A is expressed in the same periodic pattern there is a high level of expression in the posterior com- partment coextensive with the en stripe Then it decreases gradually along the anterior compartment to reach the lowest level just anterior to the next en stripe (Fig. 1A).

To know when the main features of abd-A expression become established, we used the whole mount in s~tu method (Tautz and Pfelfle, 1989) to study abd-A tran- scription from its onset early in embryogenesls. This was carried out m wlldtype and Abd-B mutant embryos because in the latter there ~s an expansion of the abd-A protein domain (Macias et al., 1990), and it is of interest to analyze how this phenomenon is reflected at the early transcriptional level

In the wlldtype, abd-A RNA is first expressed during gastrulatlon (stage 6), in the region corresponding to parasegments 7 to 13 according to the poslt~on of the label with respect to the total egg length (Fig 1B) Originally, abd-A label is approximately uniform along the domain, but by stage 7 (Fig. 1C) there are already signs of modulation, and by stage 10 the normal mtrametamerlc modulation of abd-A is already established (Fig. 1D) In the epidermis, the RNA and protein patterns are virtually the same, although there is a shght difference In the amount of protein between parasegments 7 and 8 that it is not easily discerned at the transcriptional level However, in the nerve cord there is a difference; while abd-A protein can be detected only m parasegments 7 to 13, low levels ofabd-A RNA can also be detected in parasegments 14 and 15 (Fig IF and E).

We have compared the RNA and protein patterns in Abd-B embryos' as In the wlldtype, the evolution of the RNA expression pattern follows that of the protein in all stages At germ band elongation abd-A RNA expres- sion completely fills parasegment 13 but does not extend

to parasegment 14. By germ band retraction there 1s ad- ditional transcription in part of parasegment 14. This result suggests that in Abd-B- embryos there is a sec- ondary wave of abd-A activation, which is normally prevented in the wt (see Discussion).

2 2 Expression of abd-A m aegmentatton mutants

Segment polarlt), genes Since the most obvious feature of the distribution of

abd-A product is its segmental periodicity, we have examined the possible role of some segment polaraty genes in the control ofabd-A expression. We studied the effect of alterations in the normal expression of en, naked (nkd), patched (ptc), wingless (wg) and cubltus mterruptus (ct D) genes, which are considered to play im- portant roles in the specification of larval and adult pat- terns (Ingham, 1988). Moreover, en and ci D encode regulatory proteins (transcription factors) which show complementary &strlbutlons, posterior and anterior compartments, respectwely, in embryos and lmaglnal discs Therefore, there was the possibility they have parallel effects on abd-A transcription.

engralled The gene en appears as a prime candidate because the region of h~gh level of abd-A product corn-

A

B

$,13 e n -

Fig 2 Effect of engratled on abd-A expression In the wlldtype (A), the tracheal pits (arrowheads), which belong to the anterior compartment, exhibit intermediate level of label The most densely stained region corresponds to the posterior compartments In parasegment 13 (arrow) most of the label corresponds to the posterior compartment, as indicated by the solid line In enIKmutant embryos (B), the posterior compartments show a low level expression, indicated by the fact that the highest mark corresponds to the tracheal p~ts Also, near-

ly all the label of parasegment 13 is lacking

I~ 4 Ma{ta~ et a/ M e J u m t v m o/ Development 46 (1994; 15 25

ctdes with the en stapes (Macias et a l , 1990) We find lhat m en- mutant embryos there is a reduced level of ahd-4 protein (Fig 2B) in the postertor compartments , although there ~s httle or no alteration in the anterior

ones The hmlts of the expresston d o m a m are not rood> fled After germ band retractmn, ahd-A expression becomes umform along the domain, though the level of expression remains lower than m wtldtype Because the

)

B B

C D

wg

en

WT

wg

en en en' en

ptc

t lg 3 Lltcct ol en o '~erexpresslon on abel t express ion pa t t e rn A) Latera l ,,~e',', ol a ,,rage l I /h~p70-en e m b r y o shov, m g high and u m t o r m a&L I

e\pre, ,s lon 3 h af ter heat m d u c n o n N o u c e tha t unhke m the ~ f l d D p e (see Figs I and 2), p a r a s e g m c n t 13 is s t rongly labeled B) Ventral ~]e~ ol

a s lmdar e m b r y o to show that m a d d i u o n to the s t rong label m 13 ahd-A pro te in is now present m par t o f p a r a s e g m e n t 14 (ASpl Fhese embryos

were doub ly labeled with ant l -en (blue) and antl-ahd-4 (b rown) an t ibod ies

Fig 4 Effect o f p a u h e d m u t a n o n s on ahd-4 express ion (A) Single s ta in ing ,aHh antl-ahd-4 a n t i b o d y of a pt~ e m b r y o showing a h e r n a t m g blocks

of hagh and low ah~# 4 express ion Pa ra segmen t s 7 a n d I ~, are ind ica ted (B) Doub le s ta in ing for en and ahd- 4 ol a pt~ - embr3,o In pt~ u m t , m o n s

there is an ec toDc en str ipe m the middle ot each segment and w~ express ion is e x p a n d e d an te r io r ly I M a r t m e T - A n a s et a l , 1988) N o u c e that the

zone of high ahd-A express ion c o r r e s p o n d s to the regxon defined b> the no rma l en s m p e (think a r row) and the next pos te r ior ectoplc en str ipe (dun

arrov,) The zone o f low ahJ-4 expres ,qon extends f rom the ectop~c en ,,tripe to the next pos te r io r normal str ipe and coincides with the ~cglon ,~here ~t'z xs expanded O u r m t e r p r e t , m o n of th~s result ~s i l lus t ra ted m 1( ) a n d (l)) The ectop~c en str ipe ~s m re,~ersed polanD, wHh respcct to the no rma l

s m p e , ,rod as a consequence the ahd- 4 m t r a m e t a m e r l c g rad ien t ~s also reversed This s l tuaUon genera tes within each metamere two areas v,~th dd'-

ferent le,,eN of ahd- 4 p roduc t

A Macias et al / Mechamsms o[ Development 46 (1994) 15-25 19

amount of abd-A product is normally very high m the posterior compartments, this result suggests that en may act increasing transcription of abd-A, though it is not reqmred for the original activation

We have checked the possible enhancing role of en

using a genetic construction (Poole and Kornberg, 1988) in which the en product is driven by the hsp70 promoter. This gives rise to generalized en actlwty all over the body. After 30 min heat shock given to embryos of 3 h of age, we find two significant differences in abd-A

expression with respect to the normal situation First, the intrametameric modulation disappears, the para- segments that normally contain abd-A product show a uniform and high level of antigen (Fig. 3A), indicating a strong activating effect of the en protein even in the anterior compartments of the abd-A domain where it is normally not present. Nevertheless, the anterior border of abd-A expression is not modified, thus defining a sharp limit to the activating activity of en

The second difference is that, in contrast with the observed in the anterior border of the domain, there is an alteration of the posterior one, that now extends to parasegment 14, as cells of A8p appear labeled (Fig 3B). Clearly there are some cells beyond the normal posterior limit of the abd-A domain which are sensmve to the activating properties of the en product, thus in- dicating differential requirements at the two ends of the domain.

patched The expression of abd-A in ptc- embryos is illustrated in Fig. 4A In contrast to the graded wfldtype expression (Macias et a l , 1990; Karch et al., 1990; Fig. 1A), each metamere can be divided in two zones of abd-

A expressmn' one with high, and other with low level of product. This is probably a consequence of the effect of ptc mutants on en expression, that produces an ectoplc en stripe in the anterior compartment (Martlnez-Arlas et al., 1988). Double staining for abd-A and en (4B) In- dicates that the region of high expression corresponds to the anterior part of the parasegment and is defined by the normal and ectoplc en stripes. The posterior part of the parasegment, extending from the ectoplc en stripe to the border of the next parasegment is the region where wg is expanded (Martinez-Arlas et al., 1988) and shows low abd-A expression. This expression pattern indicates an Inversion in the gradient ofabd-A expression (see Fig 4C and D) within each ptc metamere. Normally, the cell just anterior to the normal en stripe shows low, and the cell immediately posterior a high, abd-A levels. This is unaltered In ptc mutants, but with respect to the ectoplc en stripe the situation is the reverse (Fig 4D), the ante- rior cell has high, and the posterior low, abd-A level. Thus, the ectoplc en stripe generates an reversion in the normal polarity, also reflected in the larval epidermal pattern, which shows a mirror image duplication (Martinez-Arlas et al., 1988, Hooper and Scott, 1989)

The role o fp t c can also be assayed by overexpressmg

A

f

B nkd

wg"-

Fig 5 abd-A expression m naked (A) and wmgles~ (B) mutant em- bryos In A) there are alternating blocks of high and low label This probably reflects the expansion of the en domain m nkd mutant em- bryos (Martmez-Anas et al. 1988) In B) most of the mtrametamenc gradient disappears and there )s a approximately uniform, low expres-

sion level

the product using a hsp70-ptc fusion gene (Sampedro and Guerrero, 1991) In spite of the clear effect o f p t c -

mutations shown above, we find that unrestricted expression o f p t c does not alter the normal distribution of abd-A product In these embryos, en expression also remains unaltered However, heat induction ofp tc prod- uct in ptc- embryos restores the wlldtype abd-A pat- tern, as well as the normal en pattern These results clearly suggest that the effect of ptc mutants on abd-A

expression ts not direct but mediated by its effect on en naked In nkd mutants, abd-A is expressed m broad

bands (Fig 5A) resembling those observed in the case of ptc Double staining with en shows that the area of high expression corresponds cell by cell with high en expres- sion, that In the case of nkd mutants is expanded and occupies about half parasegment (Martinez-Arlas et al., 1988). We observe that the region of low abd-A expres- sion 1S coincident with the wg domain.

wingless The expression of abd-A in wg- embryos was studied by double labeling with anti-en and antl- abd-A antibodies in some experiments, and with single antl-abd-A in others. The loss of e n activity in wg- em- bryos (Martinez-Arlas et a l , 1988) allowed us to identify them abd-A label appears at the same time as in the wfldtype, but there is little parasegmental modulation

20 4 Ma~las el al ,'bledlantwns ol Development 46 ¢ 1994~ 15 25

(Fig 5B), the ahd-A product is more uniformly distri- buted m the entire domain than m the wlldtype and the lnequalltmS observed are not periodical . After the germ band shortens, the amount of p roduc t decreases in the epidermal cells but increases in the matur ing nerve cord. At this time the morpho logy of the embryos becomes very abnormal

The role o f wmgles,s was further studied by using a hsp70-irg gene generously provided by Dr P A, Lawrence Al though the effect is variable, p robab ly due to differences m the response to the heat shock, we frequently observe embryos with abnormal ahd-A

expression; Instead of the normal gradient of product there are two well defined areas, one with high and the other with low level of product This pat tern closely resembles that described for nkd mutants . Double stain for abd-A and en shows that as in nkd mutants , the en stripe has b roadened because of the incorpora t ion of cells poster ior to the normal en domain It appears that the main consequence of the ectoplc expression of wg is an extension of the en domain which m turn results m an elevation of the abd-A levels There is no effect of the ectoplc presence o f wg product m the poster ior part of the parasegment We also examined ahd-A expression m w,k'- en- embryos, which is very similar to that seen in i ,~- embryos a low, uniform level of ahd-4 All together, these experiments indicate that most or all the effect of w~, is also mediated by its effect on en actwlt?

ubtlu,s mterruplu,s We had no unambmguous wa~¢ to identify the ci D homozygous embryos, but till the em- bryos m the balanced heterozygous stock appear to ha,~e a normal expression ofahd-A, al though in some we find a part ial loss of the mt rametamer lc pat tern These results suggest that ct D does not have a slgmficant role on ahd-A expression

B

e v e o d d

big 6 Alterations ol ahd-,l cxpre~',lon m e~c mutd tums &) Single

staining of ahd-4 i n ( , ru RI¢ embryo 1 he extent ol the ahd- t domain

~s not altered, but the distribution ol tile product is abnormal , the nor-

mal m t r a m e t a m e n c modula t ion ~s lacking and the product p~ prcscnl

m lov, uniform amoun t along the domain B) l )oublc staining Iol ~ H

and ahd-A in the double mutan t combina t ion ~,~, . d d lhc hal-

rower stripes correspond to en bands HIe part]dl icMorat]on o[ tilt_' ~'rt

expression m the double mutan t combmdt lon re,,ults m rill elevation ol

the a&g,4 expre~.smn le,,eb, illustrating the strong lnllu¢iKc ol ~ tl on

ahd-4 expression and also that c~e is not required to ,M~le,.e high Icx-

sis ol ahd- 1 exprm, su',n

Pair-ride ,genes

The pair-rule genes estabhsh the link between the gap and the homeotlc genes and some of them, in par t icular &slu tarazu lJtz) and even-skipped (eve), act as t ranscrip-

tlonal act ivators and play a critical role in establ ishing en expression In the case of / t . : , there also is evidence (Muller and Blenz, 1992, Qlan et a l , 1993) that it direct- ly affects Uhx t ranscr ipt ion We have examined the effects on ahd-A expression of a l tera t ions m the function of eve, /tz, odd-skipped, hairy, paired, and runl

even-s'klpped Homozygous mutan t larvae for the null allele eve ~'~3 differentiate a lawn of dentlcles and show no sign of segmentat ion (Nussle ln-Volhard et a l , 1985). Besides, they exhibit vir tual ly no en ac twlD (Mac- Donald, et a l . 1986) The level of abd-A expression m these embryos is low (Fig 6A), but, as far as l! can be ascertained, restricted to the normal domain Thus, abd- A is act ivated to basal levels in the absence of eve func- tion, but subsequent increase of ahd-A activity is prevented Since there is no en activity in el'e- mutants,

this observat ion suppor ts the conclusion that en is not necessary for the original ac twat lon of ahd-A As it was not possible from these exper iments to judge whether the effect of eve ts direct or it is media ted by other fac- tors hke en or wg or others, we studied abd-A expression m the double mutan t combina t ion odd-skipped, eve Rl~

in which en activity is par t ia l ly restored ( D l N a r d o and O 'Far re l l , 1987). In these embryos , abd-A expression is

higher and much more normal than in previous case (Fig. 6B), demons t ra t ing that the function of eve is not an essential for abd-A and that its effect is media ted by at least odd and en. The low expression of ahd-A in eve

mutants contrasts with that of Ubx which becomes very high (Mar t lnez-Ar las and White, 1988). Possibly, these two facts are related and the high level o f Ubx is in part a consequence of the low expression ofahd-A which nor- mally down-regulates Uhx (Struhl and White, 1985)

A MaHa9 et al /Mectlanl~m,s o/Del'elopment 46 (1994) 15-25 21

fusht tarazu In f tz null mutants, the original parasegmental chain 1-15 is modified and a chain of d o u b l e m e t a m e r e u m t s l + 2 , 3 + 4 , , 1 3 + 14~sform- ed. Each umt dafferentlates in each case the pattern of the anterxor parasegment. As a result, the f tz- embryos appear to lack even-numbered segments (see Lawrence, 1992). These embryos lack en expressmn stripes of even parasegments (Howard and Ingham, 1986)

Wxth respect to abd-A expression, f tz w2° embryos ex- hibit a double segment periodicity. It is expressed m the 7 + 8, 9 + 10, 11 + 12 double parasegments and in a small portion of the 13 + 14 unit. The lntrametamerlc pattern resembles the wfldtype one, showing the same gradient of expressmn observed m the normal metameres, but along the double parasegment umt. The first labeled metamere, formed by parasegments 7 and 8

A

B A~p B

lUII

C

Z ~ 6 + 7 ~ 8~" ~ prd-

r ' l ,~T'tZ

Fig 7 Effects of alterations in the/usht tarazu function on abd-A expression In (A) the loss o f / t : activity produces double parasegmental units, 7 + 8, 9 + 10, etc The ahd-A domain is not altered, but the 7 + 8 unit has a low expression level resembhng the level normally found m parasegment 7 (B) and (C) The overexpresslon o f / t z (HS/tz) results in embryos in which there is a normal set of parasegments (B), or double parasegmental units 7 + 8, 9 + 10, etc (C), just as an f t z - embryos In both cases there is a high level of abd-A expression which ehmlnates the normal gradmnt of product Also, the abd-A protein is found in parasegment 14 (A8p), where normally abd-A Is not active (B) Notice that the 7 + 8 unit in (C)

exhibits a high and uniform level of product in contrast with the same unit m f t z - embryos

Fig 8 ahd-A (brown) and en (blue) expression in runt (A) and paired (B) mutants In (A) the dlStrlbutmn of ahd-A product is not detectably graded but appears In alternating blocks, defined by the en stripes, and possessing high or low amounts of product As m the case o fp t c mutants (see Fig 4) this expression pattern can be explained by reversed polarity of alternate en stripes that generate a reversed gradient of abd-A expression In (B) the prd mutant embryo lacks the en stripe number 7, which normally marks the anterior border ofabd-A expression Yet, abd-A is activated there, ~f at a low level A gradient of expression can also be noticed in the region corresponding to parasegment 7, and also In the double metamerlc

units

22 ! ¢la~Ja~ et al Me, barnums ol Deveh~pment 46 (1994) 15-25

shows (Fig 7A) the lower level of expression correspon- ding to parasegment 7 in the wlldtype

The role o f / t z was also assayed in an complementary experiment, in which t h e / t z product was overexpressed using an hap70-/tz fusion gene (Struhl, 1985) The mor- phological effectb of this overexpresslon have been described (Struhl, 1985, Ish-Horowlcz et a l , 1989) heat shocked embryos show a pair-rule phenotype, but the identity of the double metamere units is al tered with respect to t t z - embryos due to a different pat tern of

homeotlc gene expression ( Ish-Horowlcz et a l , 1989) The effect on ahd-A expression is i l lustrated in Fig 7B and C we observe abnormal abd-A expression pat terns that can be divided in two classes. (1) embryos that at

the extended germ band stage have a normal number of metameres, but showing elevated levels of ahd-A prod- uct within the domain Besides, the ahd-A domain is expanded posteriorly, occupying most of the A8a com- par tment and part of A8p (parasegment 14) Because of the high levels of abd-A, the normal me tamenc gradient is obscured (2) embryos exhibi t ing double me tamenc units As lndmated by the staining, the double units were the same as in l tz- embryos, 1 + 2, , 7 + 8, 9 + 10, I1 + 12, 13 + 14 parasegments , but unhke l tz- , the composi te 7+ 8 metamere exhibits a high amount ofahd-

,4 product The amount of product in the 13 + 14 unit

is greater than in / t z - embryos These experiments suggest t h a t / t z acts as a trans acti-

vator of abd-A act lwty As ~t has been shown t h a t / t z overexpresslon produces ectoplc activity of en (lsh- Horowlcz et a l . 1989), we have tested whether this role lb mediated by en To this end we overexpressed llz m

en- embryos and observed a similar increase m abd-A

expression levels, demons t ra t ing that at least part of the

enhancing effect of ttz is not mediated by en punt In run mutant embryos several inetameres are

deleted and the remaining often show mir ror image duphcat lons (Gergen and Wleschaus. 1986) The

wfldtype function of run is necessary, for the normal striped pat tern of lt: and eve ( lngham and Gergen, 1988) In run mutants the normal en expression Is modi- fied (Mar t inez-Arlas and White. 1988) the en s tapes are

abnormal ly d l smbu ted , some are closer and others fur- ther apar t than in the wddtype. We have conf i rmed this and also found that the en stripes frequentl~ fuse, especmlly on the lateral sides The normal abd-A expres- sxon is also modif ied in run mutant embryos, ahd-A label appears in al ternat ing blocks showing high or low ex- pression (Fig 8A) We have compared en and abd-A protein dis t r ibut ions by simple and double labeling As il lustrated in Fig 8A, the blocks of high ahd-A expres- sion are defined by two en stripes, but include areas with no en activity These results suggest that m run mutants , in addi t ion to an al terat ion In the d l sposmon of the en

stripes, there is a change m polari ty of a l ternat ing btnpes, which is reflected in the ah~gA protem dls t r lbu-

tlon This change in the polar i ty of abd-A expression, and presumably of other homeot lc genes, is p robably re-

sponsible of the mir ror image dupl icat ions found (Gergen and Wleschaus, 1986) m the larval epidermal pat terns of run mutants

halo' In h m u t a n t s / t z and en expressions are altered (Howard and Ingham, 1986, D1Nardo and O 'Far re l l , 1987) There are only seven en stripes and these are broader than in wlldtype Also, the p o s m o n m g of the en stripes is not regular, the spacing between them varies

The expression of abd-A Is also modif ied with respect to the wxldtype, and in general t\~llows the effect on en, the areas of high en expression coincide with those of ah~LA We sometimes observe abd-A label in the en stripe

corresponding to ASp, which in the wlldtype contains no detectable abd-A product This might be due to the expansion of fiz stripes m h mutants (Howard and Ingham, 1986)

pawed In prd mutants parasegments are grouped m double metamere units, 2 + 3 , , 6 + 7 , 8 + 9 , 12 + 13, 14 + 15, in different frame from those o f / t z mutants The pat tern differentiated by each unit is a composi te of the pamc tpa t l ng parasegments (D1Nardo and O 'Far re l l , 1987) The en stripes cor responding to odd parasegments are lacking We find that ahd-A is

expressed in the same domain as m the wlldtype, but

with a different pat tern The anter ior limit is m the inxd- dle of the 6 + 7 unit, in the region cor responding to the absent 7th en stripe (Fig, 8B), a l though the level of expression is much lower than in normal parasegxnent 7, p robably due to the lack of the act ivat ing effect of cn In the other double umts of the domain the ah~gA pro- teln is dis t r ibuted following a gradient resembhng that of the wlldtype but extending to the equivalent of two parasegments (Fig 8B)

3. Discussion

3 1 Factors governmg the modulation o/ahd-A a~tlvltv

In this paper we have examined some of the genetic factors governing abd-A expression We have tested the role of some pair-rule (eve, fiz, prd, run, h)and some seg- ment polari ty (en, wg, nkd, ptc and c~ I)) genes on abd-A

expression The first conclusion is that none of these genes is

essential for abd-A original act ivat ion, the normal domain of abd-A is establ ished in their absence This indicates that pr ior to the pair-rule and segment polar i ty genes there are other factors (prime candMates are the gap genes, Sfinchez-Herrero pers c o m m u n ) whmh estabhsh the lnitml domain The roles of the segment polar i ty and pair-rule genes appear to be related with the subsequent e labora t ion o f the initial abd-A pat tern local differences of expression within metameres, and mtermetamer lc differences.

A Macias et al / Me~hamsm~ of Development 46 (1994) 15-25 23

Of the segment polarity genes, a major role in this process corresponds to engraded, which acts as a trans activator of abd-A: any modification in en expression is paralleled by abd-A expression. The effects of nkd and ptc are most probably mediated by their respective effects on en. Also, the effect of some pair-rule genes like eve, runt or hairy is probably mediated by en. Given the nature of the product (Fjose et ai., 1985), these observa- tions strongly suggest that en functions as a transcrip- tional regulator of abd-A. Thus, it is probably responsible for the hlgh abd-A expression levels normal- ly found in posterior compartments.

However, the normal graded expression in the ante- rlor compartments demands specific factors to this com- partment In wg- embryos this anterior gradient disappears, suggesting that wg might be connected with this process. These embryos show an uniform low level of abd-A in the whole domain, including the posterior compartments and similar to that seen m en-, wg- embryos. Part of the effect is probably due to the loss of en activity in wg- mutants (DiNardo et al., 1988, Martinez-Arias et al., 1988), though the partial loss of the anterior gradient must have another explanation The gene Cl D, encoding a transcriptional factor specific of anterior compartments (Orenlc et al., 1990) does not seem to affect abd-A expression

The above observations suggest tlle segment polarity genes, and especmlly en and wg, may have a role in the elaboration and maintenance of homeotlc gene activity. wg activity is required for the autocatalysls of Ubx m the visceral mesoderm (Thuranger and Blenz, 1993; Thur- inger et a l , 1993) and of Deformed in the epidermis (Gonzalez-Reyes and Morata, 1992), and en negatively regulates Ubx activity (Martinez-Arlas and White, 1988). The fact that segment polarity genes are active throughout the embryonic and larval periods supports this suggestion.

Among the pair-rule genes tested, only f tz appears to have a significant role in establishing lntermetamerlc differences in abd-A expression, in f t z - embryos the double metamere unit 7 + 8 contains low levels of abd- A protein, whereas in hsp70-ftz embryos the same umt has higher levels. Since in the wlldtype there is a differ- ence in the protein levels between parasegments 7 and 8, this suggests that the ftz product is responsible for this difference by increasing the level of abd-A activity. Although this effect may m part be mediated by en, as overexpresslon o f f t z produces an expansion of the en domain (Ish-Horowlcz et a l , 1989), we observe high lev- els of abd-A product in en-, hsp70-ftz embryos, ruling out en as the sole contributor to the increase in abd-A levels. As there is recent evidence thatftz is a direct acti- vator of Ubx transcription (Muller and Bienz, 1992), it is likely that it performs a similar function on abd-A.

One aspect worth mentioning of the enhancing role of ftz is that, like in the case of en, it is strictly hmlted by

the anterior border domain in parasegment 7, but not by the posterior border, as f tz overexpresslon gives rise to abd-A expression in cells of parasegment 14 All the cells of the parasegment 7 to 14 region are sensitive to the enhancing effect of ftz, including those which do not normally have f tz function

3 2 A second wave oJ abd-A actlvatton

In Abd-B- embryos the initial transcriptional domain extends from parasegment 7 to 13, but, unlike the wlldtype embryos, by germ band retraction it expands to parasegment 14 and part of 15 This reflects the ex- istence of a late event of abd-A act~vatlon which is nor- mally suppressed by the Abd-B product. Because it takes place at a nine when maternal and gap genes are no longer present, it must require a set of activating factors different from those involved with early activation

There is increasing evidence that homeotlc genes may have several rounds of activation during embryogenesls. Recently, we have reported (Pelaz et a l , 1993) a second- ary event of activation for the gene Sex combs reduced (Scr), in the absence of the BX-C genes, Scr becomes activated in the posterior compartments of thoracic and abdominal segments Interestingly, the late activation time ofabd-A occurs at the same time as that of Scr, sug- gesting the posslbihty that the same genetic mechanism ~s involved in the two cases

3 3 The 'mtttal' and the 'elaborated' expression domains

of abd-A

Our results permit to dlstlngmsh at least two aspects In the evolution of the abd-A expression domain. First, there is the 'initial' domain, probably specified by the activities of very early acting genes like the maternal and gap genes This extends from parasegments 7 to 13 and IS defined by parasegmental borders at the anterior and posterior ends, as indicated by the RNA expression m Abd-B- mutants after germ band elongation, which extends exactly to the lntersectmn between para- segments 13 and 14 The original levels of expression are low and uniform as illustrated by the early RNA expres- sion We do not know the molecular events underlying this process, but probably this early activation includes the 'opening' of cts-regulatory sequences which become accessible to transcriptional factors like those encoded by en or f iz

This initial situation is subsequently altered, and by stage 10 the final, 'elaborated', pattern has been established. There are at least three modifications' (1) the intrametamerlc modulation. The gene en is a major contributor to this phenomenon, and responsible for the high abd-A levels in posterior compartments Other genes like wg may be connected with the modulation within the anterior compartments, (2) the differences in

24 4 Ma¢ia,s et a l / ~e(hamsms of Developmenl 46 ~ 1994) 15-2~

the levels of abd-A product between parasegments 7 and 8 to 12. Paar-rule genes hke f tz probably contribute to this differences (3) the loss of abd-A expression in part of parasegment 13. The factor responsible for this modl- ficataon is the product of Abd-B; in its absence all the cells in A8a exhibit high levels of abd-A (Maclas et a l . 1990).

Superimposed with these modifications there also ~s the second wave of abd-A activation in parasegments 14 and 15 This is normally suppressed by Abd-B so ~t is not detected in wt embryos Nevertheless, ~t generates a condition in the abd-A gene which, even in the presence of the repressing Abd-B product• becomes sensltwe to the enhancing effects of en or / t z ~f• as m the heat shock experiments, these genes are activated those para- segments. This second round of activation may be responsible for the presence of abd-A RNA m parasegments 14 and 15 in the nerve cord of the wt. It is possible that the Abd-B product is not as efficient in the nerve cord as in the epidermis to suppress ahd-A transcriptionally

4. Experimental procedures

4 1. Mutations, chromosomes and heat shock treatments

The following mutations have been used: segment polarity mutaUons: en IK, ptc 1Nl°8 (Nfisslem-Volhard et al., 1984), wg CXC4 (Baker, 1987), nkd 7E89 (Jurgens et al., 1984) and ct ° (Orenlc et al., 1987) Pair-rule mutations:

f tz we° (Jurgens et al., 1984), eve Rl3 (Nussleln-Volhard et al., 1985), prd 24517 (Ntisslem-Volhard et al., 1984), runt Yc (Nfisslein-Volhard and Wleschaus, 1980) and Df(3L) h ~22 (Ingham et al., 1985) Homeotlc mutations: Abd-B Ml (Sanchez-Herrero et al., 1985). Heat shock lines' they all use the hsp70 promoter linked to the coding region of the genem question. For further details consult the original papers. The hsp70-en construct is described m Poole and Kornberg, 1988, hsp70-ftz in Struhl, 1985 and hsp70-ptc in Sampedro and Guerrero 1991.

Except for the ct ~ stock, balanced with the M(4) spa p°l chromosome and in which we had no indepen- dent way to distlngmsh the homozygous mutant em- bryos from the heterozygous ones, m all other cases the mutant chromosome was balanced with either a Cy-O hb-~3-gal or a TM3 hb-~-gal chromosome, so that homozygous embryos could be readily distinguished by a double staining

The heat shock treatments varied according to the features of the different heat shock hnes, but the usual protocol was a 30-mm heat shock given to embryos of 3-4 h of age. They were then allowed to develop for 2 or 3 h before fixing.

4 2 Stammg procedure,s

Single and double antibody staining procedures were as described in Macias et al • 1990 The polyclonal antl- abd-A antibody was described in Macias el al • 1990, lhe monoclonal ant~-en and ant~-Uhx ant~bo&es were described m Patel et a l , 1989 and White and Wilcox, 1984, respectively Whole mount m sltu as m Taut7 ,tnd Pflelfle, 1989

Acknowledgments

We thank Isabel Guerrero, Peter Lawrence, Jawer Sampedro, and Ernesto S~inchez-Herrero for their com- ments and help We also thank Peter Lawrence, David Ish-Horowlcz and Tom Kornberg for heat shock stocks This work was supported by a grant from the Dlreccl6n General de Investigacl6n Clentifica y T6cntca and by an lnStltUc:onal grant from the Fundaclon Ram6n Areces Ana Maclas is supported by fellowship from the D~rec- cl6n General de Investigaclon Clentifica y T&mca and Soraya Pelaz from a fellowship from the Basque Auto- nomous Government

References

Akam, M (1987) Development I1)1, 1-22 Baker, N (1987) EMBO J 6, 1765-1773 Blenz, M and Tremml, G (1988) Nature 3~3, 576-578 Bustuna, A, Casanova, J , Sanchez-Herrero, k , Gonz,ilez R and

Morata, G (1989) Development 107, 575-583 Choumard, S and Kaufman, T C (1991} Development 11

1267-1280 Cumberledge. S Zaratzlan. A and Sakon3u, S (1980} Proc Nail

Acad Scl 87, 3259-3263 DxNardo, S and O'Farrell, P (1987) Genes De,,, 1 1212-1225 DiNardo. S, Sher, E, Heemskerk-Jongens, J , Kassls, J and O Far -

rell, P (1988) Nature 332, 604-609 Fjose, A, McGmms, W and Gehrmg, WJ (1985) Nature ~13,

284-289

Gergen, J and Wleschaus. E (1986) Cell 45, 289-299 Gonz~lez-Reyes, A and Morata, G (1992) Development 116

1059-1068 Hafen, E, Levme, M and Gehrmg, W J (1984) Nature 3/)7,287-289 Hooper, J and Scott, M (1989) Cell 59, 751-765 Howard, K and Ingham, P (1986) Cell 44, 949-957 Ingham, P (1988) Nature 335, 25-34 Ingham, P and Gergen, P (1988) Development 104, 51-60 lngham, P, Howard, K and lsh-Horowlcz, D (1985) Nature ~18,

439-445 Ingham, P and Martlnez-Arlas, A (1986) Nature 324 592-597 Irvane, K D, Botas, J , Jha, S, Mann, R and Hogness, D (199~) De-

velopment 117, 387-399 lsh-Horowlcz, D Plchm, S M lngham, P W and Gyurko~lcs (1989)

Cell 57, 223-232 Jack, T and McGmms, W (1990) EMBO J 9, 1187-1198 Jurgens, G (1985) Nature 316, 153-155 Jurgens, G, Wleschaus, E, Nusslem-Volhard, C and Kludmg, H

(1984) Roux' Arch 93, 283-295 Karch, F, Bender, W and Welffenbach, B (1990) Genes Dev 4,

1573-1587

A Macia~ et al /Mecham~m~ ¢~f Development 46 (1994) 15-25 25

Karch, F , Welffenbach, B, Pelfer, M. Bender, W, Duncan, I , Celmker, S, Crosby, M and Lewis, E (1985) Cell 43, 81-96

Kuzlora, M and McGmms, W (1988) Cell 55, 477-485 Lawrence, PA (1992) The Making of a Fly Blackwell Scientific

Pubhcatlons, Oxford Macdonald, P, Ingham, P and Struhl, G (1986) Cell 47, 721-734 Mac/as, A , Casanova, J and Morata, G (1990) Development 110,

1197-1207 Martinez-Anas, A , Baker, N and Ingham, P (1988) Development

103, 157-170 Martinez-Anas, A and White, R (1988) Development 102, 325-338 Muller, J and Blenz, M (1992) EMBO J 11, 3653-3661 Nusslem-Volhard, C, Kludmg, H and Jurgens, G (1985) Cold Sprmg

Harbor Syrup Quant Blol 50, 145-154 Nusslem-Volhard, C and Wleschaus, E (1980) Nature 287, 795-801 Nusslem-Volhard, C, Wleschaus, E and Kludmg, H (1984) Roux"

Arch 193, 267-282 Oremc, T , Chldsey J and Holgreem, R (1987) Dev Blol 124, 50-56 Orenlc, T S , Kroll, K and Holmgreen, R (1990) Genes Dev 4,

1058-1073

Paro, R and Hogness, D (1991)Proc Natl Acad Scl 88, 263-267 Patel, N, Martin-Blanco, E, Coleman, K, Poole, S, Elhs, M, Korn-

berg, T and Goodman, C (1989) Cell 58, 955-968 Pelaz, S, Urqula, N and Morata, G (1993) Development 117,

917-923 Poole, S and Kornberg, T (1988) Development 104, 85-94 Qlan, S, Capovdla, M and PIrrotta, V (1993) EMBO J 12,

3865-3877 Sampedro, J and Guerrero, ! (1991) Nature 353, 187-190 Sdnchez-Herrero, E, Vernos, I , Marco, R and Morata, G (1985)

Nature, 313, 108-113 Struhl, G (1985) Nature 318, 677-680 Struhl, G and White, R (1985) Cell 43, 507-519 Tautz, D and Pfelfle, C (1989) Chromosoma 98, 81-85 Thurmger, F and Blenz, M (1993) Proc Natl Acad Scl 90,

3899-3903 Thurmger, F , Cohen, S M and Blenz, M (1993) EMBO J 12, White, R and Lehmann, R (1986) Cell 47, 311-321 White, R and Wdcox, M (1984) Cell 39, 163-171