Gradients of Krüppel and knirps gene products direct pair-rule gene stripe patterning in the posterior region of the drosophila embryo

Cell, Vol. 61, 309-317,

April 20, 1990, Copyright

0 1990 by Cell Press

Gradients of Kriippel and knirps Gene Products Direct Pair-Rule Gene Stripe Patterning in the Posterior Region of the Drosophila Embryo Michael J. Pankratz,’ Eveline Seifert,t Nicole Gerwin,’ Bettina Billi,’ Ulrich Nauber, and Herbert Jlckle” * lnstitut fur Genetik und Mikrobiologie Maria Ward Strasse la D-8000 Munchen 19 Federal Republic of Germany tMax-Planck-lnstitut fur Entwicklungsbiologie Spemannstrasse 35/ll D-7400 Tubingen Federal Republic of Germany

Summary Abdominal segmentation of the Drosophila embryo requires the activities of the gap genes Kriippel (Kr), knirps (hi), and tailless (f/l). They control the expression of the pair-rule gene hairy (h) by activating or repressing independent cis-acting units that generate individual stripes. Kr activates stripe 5 and represses stripe 6, kni activates stripe 6 and represses stripe 7, and f/l activates stripe 7. Kr and hi proteins bind strongly to h control units that generate stripes in areas of low concentration of the respective gap gene products and weakly to those that generate stripes in areas of high gap gene expression. These results indicate that Kr and hi proteins form overlapping concentration gradients that generate the periodic pair-rule expression pattern. Introduction The body pattern of Drosophila is generated by the activities of several distinct classes of genes comprising a regulatory cascade that sequentially divides the embryo into smaller units (reviewed by Akam, 1987; Ingham, 1988). The maternal coordinate genes such as bicoid (bed), nanos (nos), and torso (tar) initially organize the embryo into the anterior, the posterior, and the terminal domains, respectively (Ntisslein-Volhard et al., 1987). The zygotic gap genes hunchback (hb), Kr, kni, and t/I then interact under the influence of the maternal genes to subdivide the embryo into broad regions along the anteriorposterior axis (Nusslein-Volhard et al., 1987; Lehmann, 1988). The mechanism by which the maternal coordinate genes regulate the activities of the zygotic gap genes is fundamentally different. The anterior body pattern is dependent upon the presence of the morphogen bed, which directly activates the expression of the anterior zygotic gap gene hb (Frohnhofer and Ntisslein-Volhard, 1986; Driever and Ntisslein-Volhard, 1989). It was initially assumed that the abdominal body pattern would be similarly established by the action of the posterior maternal coordinate gene nos on the posterior zygotic gap gene kni (Ni.isslein-Volhard et al., 1987). However, it was recently

shown that ectopic presence of the hb protein in the posterior region of the blastoderm embryo suppresses abdominal development (Hiilskamp et al., 1989; Struhl, 1989a). In addition, a previous study revealed that in embryos derived from nos mutant mothers, the maternally supplied hb product persists in the posterior region (Tautz, 1988). These observations suggested that hb suppresses abdominal development and that the function of nos is to remove this inhibitory effect by acting negatively on hb. This was decisively proven by demonstrating that completely normal embryos develop in the absence of nos if the maternal hb is concurrently removed (Hiilskamp et al., 1989; Irish et al., 1989; Struhl, 1989a). These results led to the view that the posterior body pattern may not require a maternal morphogenetic input and that the mechanism that actively generates the abdominal segments could be completely under the control of the zygotic genes. This view is supported by the fact that the level of expression of the posterior gap gene kni is under the direct control of the neighboring gap gene Kr (Pankratz et al., 1989) whose realm of action extends significantly into the posterior region (Wieschaus et al., 1984). Based on these findings, we proposed that the gap genes Kr and kni act in concert to initiate the process that establishes the abdominal segment pattern (Pankratz et al., 1989). Combined genetic and molecular analyses indicate that Kr and kni most likely carry out this function by directing the expression of the pair-rule genes (reviewed by Ingham, 1988). The pair-rule genes are expressed in tandemly repeating stripes in the blastoderm embryo. The patterning of all pair-rule genes examined to date is altered in embryos mutant for any of the gap genes, indicating that gap genes are involved in controlling the expression of the pair-rule genes. Because a regulatory hierarchy exists even among the pair-rule genes-primary pair-rule genes, such as h, are required for the normal expression of secondary pair-rule genes, such as fushi tarazu (ftz) (Howard and Ingham, 1986; Carroll and Scott, 1986)-the gap genes act most likely at the level of the primary pair-rule genes. In view of the overlapping protein distributions of the gap genes, it has been proposed that the relative concentrations of the gap gene products along the longitudinal axis could be important for generating the periodic pair-rule stripes (Pankratz et al., 1989; Stanojevic et al., 1989; Struhl, 1989b). In vitro studies have further shown that both hb and Kr proteins bind specifically to a &-regulatory region of the pair-rule gene even-skipped (eve) (Stanojevic et al., 1989). However, the in vivo function of these binding sites has not yet been determined. Therefore, the mechanism by which the overlapping gap gene activities actually control the pair-rule genes is unknown. Previous studies that monitored the expression patterns of the different pair-rule genes in the various gap gene mutants have shown that the number and size of the pairrule stripes are altered within the segmental primordia affected by the corresponding gap mutations (reviewed by Ingham, 1988). The interpretation of these results has

Cell 310

Figure 1. Distribution

of kni Protein and Evolution

of h Stripes

(a) and (b) Antibody staining of whole-mount embryos with polyclonal anti-kni antibody. (a) Blastoderm embryo showing the anterior and posterior domains of kni expression. (b) Slightly later stage embryo, viewed in a different focal plane, in which the anterior stripe has formed. Note in both cases that the nuclei are stained and that the staining intensity fades gradually at the borders of expression, showing a bell-shaped distribution. Orientation of the embryos is anterior left, dorsal up. (c), (d), (e), (f), (g), (h), (i), and fj) In situ hybridization of whole-mount embryos with nonradioactively labeled h DNA probe. The individual stripes appear successively. The order of appearance seems to be stripe 1, stripes 3 and 2, stripe 4, stripe 7, stripe 5, and last, stripe 6. Stripes 3 and 4 separate at about the same time that stripe 6 forms (see also Howard, 1988). The fact that the sixth h stripe is the last to form is consistent with the view that it is activated by kni, since hi is expressed after hb and Kr (see Discussion). There is also a dorsal patch of expression anterior to stripe 1 that is not discussed in this work.

been hindered, however, by the inherent limitation of analyzing the endogenous pair-rule gene expression patterns in gap mutant embryos: one cannot determine unambiguously the stripes that are affected. For example, the expression pattern of h is affected in kni mutant embryos; i.e., in the posterior region, a broad band of expression replaces stripes 6 and 7 (Ingham and Gergen, 1988). However, any number of scenarios may exist for producing this broad band, and since its stripe identity is unknown, the different mechanisms cannot be discriminated.

To overcome this technical limitation and to investigate the function of the different gap genes in regulating the individual pair-rule stripes, we first identified stripe-specific c&-acting control elements of h by the use of promoter fusion constructs that express a reporter gene in place of the endogenous h stripes in transformed embryos. We then introduced these fusion constructs into Kr, kni, and t/l mutant embryos to study how these gap genes regulate the expression of particular h stripes in the posterior region. We find that the gap genes activate or repress spe-

of Stripes in the Drosophila Embryo

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Figure 2. Identification of Distinct h Cis-Regulatory Elements (a) Restriction map of the 14 kb h upstream region required for the rescue of embryonic h phenotype. The transcribed sequence is shown by the black rectangle. The large stippled rectangle below the line indicates the region that contains all of the subfragments to which Kr protein binds strongly. S, Sall; C, Clal; R, EcoRI; E, BstEll; K, Kpnl; B, BarnHI. (b) Fragments of the h upstream region used for h-/acZ constructs, and the resulting b-gal pattern that corresponds to particular h stripes in embryos harboring these constructs. Stripe numbers in parentheses indicate low level of expression. The construct hCRK-/acZ shows a broad band in place of stripes 3 and 4 (stripe “3/4”). We have not yet unambiguously identified the fragment responsible for stripe 2. (c) Location of different stripe control elements in the h upstream region. The assignment of the different stripe elements is consistent with those based on the analyses of the various h regulatory mutants

(Howard et al., 1988).

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to the independent the h upstream odic pattern

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We show that the peri-

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Results Individual h Stripes Appear Discretely over Time h is classified as a primary pair-rule gene: it is required for the correct

patterning

of secondary

pair-rule

such as ftz, and is likely to be directly controlled genes

(Howard

and

Ingham,

1986). The final expression stripes characteristic

1986;

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of the pair-rule

Discrete Cis-Regulatory Units of h Generate Specific Stripes There exist several regulatory mutants of h that delete expression of certain stripes in the embryo (Howard et al., 1988). This suggests that the upstream regulatory region of h is composed of independent control elements necessary to generate particular stripes (Howard et al., 1988). To test whether these elements are also sufficient to drive expression of individual h stripes, we placed various DNA fragments from the upstream control region of h (Figure 2a) next to a basal heterologous promoter that had been fused to the bacterial /acZ gene (Hiromi and Gehring, 1987). These constructs (h-/acZ) were introduced into wild-type flies using P element-mediated transformation (Rubin and Spradling, 1982). For each construct, several independent transformant lines were established and the expression patterns of B-galactosidase (P-gal) were monitored in early embryos. The fusion construct hRR-/acZ directed expression of p-gal corresponding to h stripe 7 (Figure 3a; see Figure 2b for description of all constructs used). hRK-/acZ directed expression of stripe 6 and avery weak stripe 2 (Figure 3b). hKB-/acZ directed expression of stripes 1 and 5 (Figure 3c), and hRS-/acZ directed expression of only stripe 1 (Figure 3d). hSK-lacZ directed expression of stripes 2, 3, 6, and 7 and a very weak stripe 4 (Figures 3e and 3f), and NC-/acZ directed expression of stripe 3 and a weak stripe 4, which can be seen only after overstaining (Figure 39). These results demonstrate that the separate c&acting control units of the h upstream region are sufficient to drive expression of individual stripes. We then asked how the stripes in the prospective abdominal region of the embryo were regulated by Kr, kni, and t/l activities.

Carroll

genes,

by the gap and

Scott,

of h shows the seven genes (Ingham

et al.,

in which these stripes appear differs significantly (Figures lc-lj; see also Howard, 1988). Stripe 1 seems to be the first to form (Figure lc), followed by stripes 3 and 2 (Figure Id). Stripe 4 then appears abutting stripe 3 (Figure le), followed by stripe 7 (Figure If) and stripe 5 (Figure lg). Stripe 6 is the last stripe to be expressed (Figures lh and li). Concurrently, stripes 3 and 4 separate (Figures lh and li). These 1985; see Figure lj). However, the manner

Distinct Gap Gene Activities Activate or Repress Specific h Stripes in the Posterior Region The gap genes are expressed in broad overlapping domains along the longitudinal axis of the blastoderm embryo (Gaul and Jackie, 1989; Pankratz et al., 1989; Stanojevic et al., 1989). The Krprotein forms a steep asymmetric gradient, with high levels in the central region and low levels extending considerably into the posterior region (Gaul et al., 1987; Gaul and JBckle, 1989; Pankratz et al., 1989). The endogenous expression of h stripes 2-6 is altered in Kr mutant embryos (Ingham et al., 1986), indicating that Kractivity is required for proper h expression. However, as pointed out earlier, the mechanism by which Kr controls h expression is not clear, since the manner in which the individual stripes are altered is unknown. To determine how the individual h stripes were regulated by Kr activity in the posterior region of the embryo, we introduced the various h-/acZ constructs into lack-offunction Kr mutant embryos. In these mutants, k-gal expression driven by the h stripe 5 control unit disappears (Figure 4a), while p-gal expression driven by h stripe 6

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a

Figure 3. B-Gal Expression Patterns Corresponding to Particular h Stripes in Embryos Harboring Various h-/acZ Fusion Constructs

d

e

f

g

h

control unit expands and shifts slightly anteriorly (Figures 4b and 4~). This suggests that &activity is required to activate h stripe 5 expression and also to set the anterior border of h stripe 6 expression. Therefore, the broad band replacing stripes 5 and 6 in the endogenous pattern of h in Kr embryos (Ingham et al., 1986) is due in part to the disappearance of stripe 5 and the anterior expansion of stripe 6.

a

C

b

d

e

h

(a) Stripe 7 (hRR-/a@. (b) Stripe 6 (hRK-lad); this construct also expresses stripe 2 at a very low level. (c) Stripes 1 and 5 (hK&/acZ). (d) Stripe 1 (/IRS-/acZ). (e) Stripes 2, 3, 4, 6, and 7 @SK-la@; the expression of stripe 4 is very weak. (f) Same as in (e), but at an earlier stage; stripe 4 cannot be seen in this embryo. (g) Stripe 3 and a very weak stripe 4 @CC/z?‘), which is vtsible only after overstaining (data not shown). (h) Stripes 2 and 6 and what seems like a fusion of stripes 3 and 4 (hCRK-lad). The orientation of the embryos is anterior left, dorsal up. Embryos in (a), (b), (c), (d), and (f) are at blastoderm; those in (e), (g), and (h) are undergoing gastrulation.

The protein product of the posterior gap gene kni is distributed from ~27%-43% egg length (0% is posterior pole) at blastoderm (Figure la). Because the Kr protein is found from about 33%-600/o egg length (Gaul and Jackie, 1989) there is already a substantial region of visually detectable overlap between the two gap proteins. As with the Kr protein domain, the kni protein domain does not show a sharp boundary but fades at the borders, forming a bell-

Figure 4. Alterations in the D-Gal Expression Patterns Corresponding to Particular h Stripes in Different Gap Mutant Embryos The various h-lacZ fusion constructs that direct expression of f&gal corresponding to specific h stripe(s) were introduced into different gap mutant embryos. The constructs are those described and used in Figure 3. (a) Stripes 1 and 5 in Kr mutant; stripe 5 disappears. (b) Stripe 6 in Krmutant; this stripe expands anteriorly. (c) Stripes 2, 3, 4, 6, and 7 in Kf mutant; stripe 6 expands anteriorly, and a broad continuous band of expression replaces stripes 2, 3, and 4. (d) Stripe 7 in kni mutant; this stripe expands anteriorly. (e) Stripes 2, 3, 4, 6, and 7 in kni mutant; stripe 7 expands anteriorly, stripe 6 disappears (see below), stripes 2 and 3 seem relatively‘unaffected, and stripe 4 is too weak to determine. (f) Stripes 2, 3/4, and 6 in kni mutant; stripe 6 disappears and stripe 3/4 expands posteriorly. Consistent with this observation, we do not see expression of stripe 6 alone (from construct hRK-/act) in kni mutant embryos (data not shown).

(g) Stripes 2, 3, 4, 6, and 7 in f/I mutant, stripe 7 disappears and stripe 6 shifts posteriorly, while stripes 2, 3, and 4 seem unaffected. (h) Stripe 6 m r// mutant; thts stripe shafts posteriorly, consistent with what is observed in (9). Embryos In (a), (b), (c), (d), (e), and (g) are at blastoderm; embryos in (f) and (h) are undergoing gastrulation. We do not know how stripes 2, 3, and 4 are indivtdually affected by Kr activity, and we have not yet unambiguously determined the fate of stripe 5 in kni mutant embryos.

t;;eration

of Stripes in the Drosophila

Embryo

shaped distribution (Figures la and lb). Lack-of-function kni mutants exhibit pattern defects that, when projected

different gap genes on the distinct cis-regulatory units that drive the expression of individual stripes.

onto the blastoderm fate map, derive from about 22%47% egg length (Lehmann, 1988). Thus, the region affected by kni mutation and the region covered by the kni protein staining do not coincide. However, as previously observed for Kr (Gaul and Jlckle, 1989; Pankratz et al., 1989), this discrepancy could be due simply to the level of sensitivity of the immunochemical detection methods. To determine how the posterior h stripes were regulated by kni activity, the various h-lacZ constructs were introduced into kni mutant embryos. In these embryos, bgal expression driven by h control units that include stripes 6 and 7 show a broad band in place of the two posterior stripes (Figure 4e). This is also observed with the endogenous h expression pattern (Ingham and Gergen, 1988), and one cannot discern how, and which, stripes are affected. However, pgal expression driven by h stripe 7 control unit alone expands anteriorly (Figure 4d). In addition, &gal expression driven by h stripe 6 control unit alone is absent in kni mutant embryos (data not shown). To confirm this, we made a construct that expresses not only stripe 6 but in addition what appears to be a fusion of stripes 3 and 4 (hCRK-/acZ; see Figure 3h). The fused band of expression served as an internal control with which to determine the fate of the sixth stripe. As seen in Figure 4f, Dgal expression corresponding to stripe 6 disappears, while the fused stripes 3 and 4 expand posteriorly. Taken together, these findings indicate that kni is required to activate h stripe 6 and to set the anterior border of h stripe 7. Therefore, the broad band replacing stripes 6 and 7 in the endogenous h pattern in kni embryos is due to the disappearance of stripe 6 and the anterior expansion of stripe 7. As h stripe 7 lies outside the currently detectable limit of kni protein expression, these results further illustrate the long-range effect of kni activity and supports the view that kni, like Kr, forms a steep gradient in the blastoderm embryo. The terminal gap gene t/l is required for the development of the two polar regions of the embryo (Strecker et al., 1986). In t/l mutant embryos, egal expression driven by h stripe 7 control unit disappears (Figure 4g), while P-gal expression driven by h stripe 6 control unit is shifted posteriorly (Figures 4g and 4h). This finding is consistent with what is observed for endogenous h transcript pattern in f// mutants, which also shows deletion of the seventh stripe and a posterior shift of the sixth stripe (Mahoney and Lengyel, 1987). These results indicate that N/formally activates h stripe 7. However, ii is unclear how t/l controls stripe 6 since only a shift, and not a significant expansion, is seen in t// mutants. It may be that t/l does not directly affect stripe 6 and that the shift observed is due to the posterior expansion of Kr and kni expression domains in t//mutant embryos (Pankratz et al., 1989; see also the legend to Figure 6 and Discussion). Taken together, the above results show that Kr, kni, and t/l each activate or repress specific stripes along the longitudinal axis of the embryo. The periodic pattern could thus be generated by superimposing the effects of the

Binding of Kr and kni Proteins to h Upstream Region: Inverse Correlation between Binding Strength and Gap Gene Concentration All of the molecularly characterized gap gene products possess DNA binding motifs (Rosenberg et al., 1986; Tautz et al., 1987; Nauber et al., 1988). In vitro footprinting analyses have shown that hb, Kc and kniproteins can bind DNA in a sequence-specific manner (Treisman and Desplan, 1989; Stanojevic et al., 1989; Pankratz et al., 1989; Hoch and H. J., unpublished data). It has further been shown that Kr can act as a transcription regulator (Pankratz et al., 1989). It seems likely, therefore, that the gap genes directly control primary pair-rule gene patterning at the transcriptional level. We therefore screened the h upstream region for potential gap protein binding sites by immunoprecipitation (Desplan et al., 1985; Driever and Ntisslein-Volhard, 1989; Pankratz et al., 1989). A 14 kb Xho-Xba fragment (Figure 2a) that contains the regulatory information required for embryonic rescue of the h mutant phenotype (Rushlow et al., 1989) was first tested for Kr binding. As shown in Figure 5a (lanes 1 and 2), a specific set of subfragments precipitates, indicating that Kr protein binds specifically to several sites within the h upstream region. The differences in the intensities of the precipitated subfragments suggest that they contain different affinity and/or number of Kr binding sites. Surprisingly, all of the precipitated subfragments derive from a 6 kb BstEll-BamHI region (Figure 5a, lanes 3 and 4; marked by the stippled bar in Figure 2). This region does not contain the h control elements that generate the stripes found in the area of the embryo with high Kr concentration, i.e., stripes 3 and 4. To test further whether any strong Kr binding sites existed within the cis-acting unit that controlled h stripes in a region of high Kr level, we repeated the immunoprecipitation with the entire fragment that controls stripes 3 and 4 (the Clal-Clal fragment of the construct hCC-/acZ; see Figure 2). As shown in Figure 5b (lanes l-3), this fragment (marked as “E”) precipitates very weakly. In contrast, fragment “C;’which derives from a construct that regulates stripe 6 and served as a positive internal control, precipitates strongly. These results suggest that Kr protein binds weakly to h control elements that generate stripes in areas of high Kr concentration, while binding strongly to those that generate stripes in regions of low Kr concentration. We then asked whether kni would also show a similar, inverse correlation between gap protein concentration and binding strength for h control units. h stripe 6 lies in a region of the embryo with high kniprotein concentration, while h stripe 7 is located in an area of kni expression that cannot be currently detected by antibody staining. Therefore, individual h DNA fragments responsible for generating stripe 6 or stripe 7 (fragments from constructs hRK/acZ and hR/?-/acZ, respectively; see Figure 2) were tested for kni protein binding. As shown in Figure 5c, the fragment that directs expression of stripe 6 (marked as “F”)

Cell 314

a

Kr 12

Kr 34

b

Kr ---

contr

1234567

kni

Figure 5. Kr and kni Protein Binding on h Upstream Region

(a) lmmunoprecipitation of h upstream sequence with Kr protein and antibody. Lane 1: 113) I)*-E Input DNA consisting of the 14 kb Xhol-Xbal fragment (see Figure 2a) digested with Hpall and end labeled. Lane 2: Precipitated subfragments with Kr extract; note the differences in the intensities of the bands, indicating different binding strength of Kr for the various subfragments. Lane 3: Input DNA consisting of the 5.9 kb BstEll-BamHI fragment (see Figure 2a) digested with Hpall and end labeled. Lane 4: Precipitated subfragments with Kr extract; note contr kni that all of the precipitated subfragments pres4 ent in lane 2 are also present in this lane, indieating that all the sites to which Kr protein strongly binds are located within the 5.9 kb BstEll-BamHI region (denoted by the large stippled rectangle in Figure 2a). In both sets of experiments, no fragments precipitate with extracts that do not contain Kr protein (data not shown). The precipitated subfragments A, C, and D map to the 2.6 kb EcoRI-Kpnl region that controls stripe 6; subfragment B maps to the 1.2 kb EcoRI-BamHI region that controls stripe 1 (see Figure 2a). The srzes of A, B, C, and D are ~150, 390, 470. and 510 bp. respectively. The other precipitated subfragments have not yet been mapped. (b) lmmunoprecipitation of h fragment controlling stripes 3 and 4 with Kr and kni proteins. All experiments were performed with the corresponding which controls stripe 3 and 4 (marked as “E”), and antibody. Lane 1: Input DNA consisting of the intact 1.7 kb Clal-Clal fragment from hCC-/acZ, subfragment C, which derives from a region controlling stripe 6 (see above; marked as “C). The latter served as a positive internal control. Lanes 2-7: Precipitated fragments. Lane 2: Krextract (4 ~1). Lane 3: kextract (12 ~1). Note that the Clal-Clal fragment precipitates very weakly as compared wrth subfragment C. Lane 4: Control extract (12 nl) with Kr antibody. Lane 5: Control extract (12 nl) with kni antibody. Lane 6: kni extract (4 ~1). Lane 7: kni extract (12 ~1); note this time that the Clal-Clal fragment precipitates very strongly as compared with subfragment C. Therefore, Kr binds very weakly to h control elements that control the expression of stripes 3 and 4, whereas kni protein binds to this element strongly. (c) lmmunoprecipitation of h fragments controlling stripes 6 and 7 with km protein and antibody. Lane 1: Input DNA consisting of the intact 2.6 kb which controls stripe 6 (marked as “F”) and the 1.5 kb EcoRI-EcoRI fragment from /x%-/ad, which controls EcoRI-Kpnl fragment from hRK-/acZ, stripe 7 (marked as “G”). Lanes 2-5: Precipitated fragments. Lane 2: Control extract (4 ~1). Lane 3: Control extract (12 ~1). Lane 4: kni extract (4 WI). Lane 5: kni extract (12 ~1). Note that kni protein binds strongly only to the fragment that controls stripe 7.

precipitates very weakly. However, the fragment that directs expression of stripe 7 (marked as “G”) precipitates strongly. Furthermore, the fragment that controls the expression of stripes 3 and 4 (“E”), which also lie in an area of low kni protein concentration and were previously shown to precipitate very weakly with Kr protein, precipitates strongly with kni protein (Figure 5b, lanes 6 and 7; note that the lanes containing the precipitated fragments show opposite profile for Kr and kni proteins). In all of the experiments, no fragments are precipitated when neither of the gap proteins are present in the binding reaction (lanes marked “contr”). These results indicate that the kni protein binds strongly to h control units that generate stripes in an area of low kni concentration but binds weakly to those that generate stripes in regions of high kni concentration. This is analogous to what was observed for Kr Discussion Krand kni can have multiple regulatory effects on different h stripes that lie in its domain of activity: Kr is required to activate stripe 5 but delimits the anterior border of stripe 6; kni is required to activate stripe 6 but delimits the anterior border of stripe 7. These results suggest that the gradients of Kr and kni proteins each determine at least two distinct spatial boundaries along the longitudinal axis of the blastoderm embryo. In addition, the proper formation

of each of stripes 6 and 7 requires the activities of at least two gap genes. These observations, together with the data that Kr and kni can directly bind to the upstream region of h, strongly suggest that the different concentrations of Kr and kni proteins encode positional information directly decoded by the distinct cis-regulatory units of h, which direct expression of individual stripes in the posterior region of the blastoderm embryo. Before discussing the implications of these findings, we would like to point out some limitations of our experiments. First, it is difficult to assess the extent to which the P-gal stripes that are formed by the isolated h control units actually represent the authentic h stripes. Aside from the obvious difference in the stability of the b-gal and the endogenous h transcripts, there also seems to be differences in the level of expression (e.g., stripes 3 and 4 formed by the WC-/acZ construct appear to be weaker than those formed by the hSK-/acZ construct); in terms of the spatial distributions, we can only detect major deviations with our assay (e.g., the broad central band formed by the hCRKlacZ construct). For the immunoprecipitation studies, several factors could account for the differences in the intensities of the precipitated bands, including the number of binding sites, the differential binding affinities for a given site, the spacing between certain binding sites, etc. More rigorous in vitro studies will be required to discriminate between the various possibilities.

$?eration

of Stripes in the Drosophila

Embryo

Maternal bed Gradient vs. Zygotic Kr and kni Gradients The ability of the different control elements of h to respond to spatial cues set up by Kr and kni is formally analogous to the postulated ability of different genes to respond to the spatial cues established by the maternal morphogen bcd(Driever et al., 1989; Struhl et al., 1989). The bcdgradient most likely generates contiguous domains of subordinate gene expression, thus requiring that a given target gene be able to sense a particular threshold level of the bed protein in order to be activated or repressed. This is the situation found for hb, which seems to be activated in all regions of the embryo where a critical minimum level of bed concentration exists (Driever et al., 1989; Struhl et al., 1989). In contrast, the gap genes must provide spatial cues that generate a periodic pattern throughout the segmental primordia of the embryo. To have only a single gradient provide positional values would require that the independent h control units sense and react to small concentration differences of the effector protein at intermittent spatial intervals. In addition, unlike the one-sided bcdgradient, the gradients of Krand kni are bell shaped, meaning that certain concentration values for each protein will be found in two distinct regions of the embryo. Both situations could be avoided by having an opposing gradient superimposed onto the first. This enables the specific h control units to read positional values determined by two effecters, i.e., to respond differentially to the concentrations of two proteins. Conversion of Linear Positional Information into Periodic Pattern Several potential mechanisms exist by which the overlapping gradients of Kr and kni could generate the different h stripes in the posterior region of the blastoderm embryo. We favor the view that certain threshold levels of both proteins activate or repress h transcription and that the final pattern arises from the superimposition of the separate activities of the two gap genes. The formation and behavior of the sixth h stripe can be used to illustrate this in molecular terms. We initially assumed that Kr and kni regulate h control unit 6 in two distinct manners: a certain threshold level of kni protein activates transcription, while a certain threshold level of Kr protein represses transcription (see Figure 6). If repression can override activation, then the h control unit will be activated only in a specific, narrow region of the embryo where there is sufficient level of kni for activation but insufficient level of Kr for repression. In such a system, there will be no h expression in the absence of kni activity. On the other hand, in the absence of Kr activity, h will be expressed in a much broader domain and in fact, should approximately coincide with the kniexpression domain. This is what is observed when the h control unit that drives expression of stripe 6 is placed into knior Kr mutant embryos: stripe 6 is absent in kni mutants but is expressed in a broad band covering -300/o43% egg length in Kr mutants, i.e., a region of kni expression. (We point out that the spatial domain of kni expression is itself not altered in Kr mutants [Pankratz et al., 19891.) This mechanism is further supported by the

OR=

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Figure 6. Generation of the Sixth h Stripe through Superimposition Distinct Gap Gene Activities

of

Kr and kni protein gradients overlap in the posterior region of the embryo. Kr activates, while kni represses, transcription from the control element that drives expression of the sixth h stripe (line with oval and square inside the four schematized nuclei). The critical factor is the different threshold levels at which the two gap proteins act. knican activate transcription only at relatively high concentrations (found in a region delimited by the stippled bar). However, Kr can repress transcription even at very low concentrations (down to the level denoted by the black arrow). This is effected through the different binding strengths that the two gap proteins have for this particular h control element: Kr binds strongly (Kr binding sites represented by ovals), whereas kni binds very weakly (kni binding sites represented by squares). This means that the kni binding site will be occupied only in a region of high kni concentration (stippled squares), while Kr binding sites will be occupied in areas of both high and low Kr concentrations (black ovals). If repression can override activation, this particular promoter element WIII then be turned on only in a region of the embryo that has sufficient levels of kni to activate, but insufficient levels of Kr to repress, transcription. As a result of the distributions of Kr and kni proteins in the blastoderm embryo, this situation would be found only in a narrow region near the posterior end, i.e., where the sixth h stripe is located (Kr is expressed at low levels down to -33% egg length, kni protein domain is visible from about 27%-43%, and the sixth h stripe is located at around 27%-33%). Given the present data, this is the minimum requirement for generating the sixth h stripe. Regulatory inputs from other sources, such as the gap genes t// and giant, or the pair-rule genes eve and runt may also be required.

manner in which the endogenous h gene is expressed: stripe 6 is the last h stripe to be activated (see Figures lg and lh), consistent with the finding that kni is the last gap gene to become expressed (Pankratz et al., 1989). This model would also explain why the control unit for h stripe 6 contains low-strength kni binding sites and high-strength Kr binding sites (see Figure 5). A low-strength kni binding site assures that activation occurs only in an area of high kni concentration; a high-strength Kr binding site assures that repression can take place even in regions of low Kr concentration. A parallel mechanism could be used to generate the seventh h stripe. A high threshold level of t/l activity would be required to activate h stripe 7, while kni activity would

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repress its expression at a low threshold level. Consistent with this formalism, the seventh stripe is absent in f/l mutant embryos, while it expands anteriorly in kni mutant embryos. Furthermore, the h control unit that directs expression of stripe 7, which lies in an area of low kni concentration, contains strong kni binding sites in comparison with the control unit that directs stripe 6, which lies in an area of high kniconcentration. A clearer understanding of the formation of the seventh h stripe must await the molecular analysis of the t/l gene. (We point out that a given h stripe need not only be activated by high levels of a gap gene product, since stripe 5, which lies in an area of relatively low Kr concentration, is activated by Kr activity.) In vitro binding studies with hb and Kr proteins have recently shown that the control element responsible for directing expression of stripe 2 of the pair-rule gene eve in an area of high hb and low Kr concentrations contains three hb and six Kr binding sites (Stanojevic et al., 1989). In contrast, the element responsible for directing eve stripe 3 in an area of low hb and high Kr concentrations contains as many as 18 hb binding sites but no highaffinity Kr sites (Stanojevic et al., 1989). These observations suggest that the formal mechanism proposed here for the generation of h stripes may apply to stripe formation of other primary pair-rule genes. Furthermore, they suggest that the overlap between hb and Krproteins in the anterior region of the embryo may be functionally equivalent to the Krand kni protein overlap in the posterior region of the embryo in terms of their effects on the pair-rule genes. Thus, a series of overlapping gradients of hb, Kr, and kni proteins, in combination with the distinct pair-rule gene control units, which are specifically activated or repressed at different concentrations of the different gap gene products, may generate a periodic pattern along the longitudinal axis of the early embryo. This initial periodicity, in turn, could be used to template additional periodic patterns by local activation and/or repression of downstream pair-rule genes. Experimental Procedures Production of kni Antibody A BamHl fragment within the 3% portion of the kni coding sequence (Nauber et al., 1988) was cloned into the Baml-ll site of the T7 pet expression vector (Studier and Moffat, 1986). Bacteria harboring this construct were induced as described (Studier and Moffat, 1986). and the crude bacterial extracts were run on an SDS-polyacrylamide gel. The specifically induced protein band with the predicted molecular weight was excised and used to immunize rabbits. In Situ Hybridization of h in Whole-Mount Embryos The whole-mount in situ hybridizations were performed as described (Tautz and Pfeifle, 1989) and were generously provided by M. Hi& kamp and D. Tautz. Germline Transformation of h-/acZ Constructs The different h upstream fragments were first cloned into the pBST vector (Stratagene) and subsequently cloned into the Kpnl-Xbal site of the vector HZ50PL, which contains the basal heat shock promoter fused to the bacterial /acZgene (Hiromi and Gehring, 1987). The fusion constructs were integrated into the Drosophila germline by P element-mediated transformation (Rubin and Spradling, 1982). Embryos harboring these constructs were stained for expression of b-gal using anti+gal antibody (Macdonald and Struhl, 1988). At least two independent transformant lines were used for all experiments.

The posterior b-gal stripes formed under the control of the distinct cis-acting h control units correspond roughly in position to the endogenous h stripes. From the in situ hybridization pattern in whole-mount embryos, stripes 5, 6, and 7 are located at m34%-39%, 28%-33%, and 20%-26% egg length, respectively. The P-gal stripes generated by the corresponding h control units for stripes 5, 6, and 7 are located at ~35%-38%, 27%-33%, and 21%-28% egg length, respectively. However, due to the very dynamic manner in which the different endogenous h stripes are formed (see Figures lc-lj) as compared with the stable expression of the p-gal stripes from the fusion constructs (due to the stability of the /acZ RNA and/or protein), any subtle differences in the amount or the position between the endogenous transcript and b-gal stripe expressions would not be detectable in our system. lmmunoprecipitation of h Fragments with Kr and kni Proteins lmmunoprecipitation was performed as described (Benson and Pirotta, 1987). Kr and kni proteins were produced using the T7 bacterial expression system (Studier and Moffat, 1986). A truncated form of the Krcontaining the DNA binding finger motif was used (Pankratz et al., 1989). Full-length kni protein was made by introducing a Ndel site at the start of the kni protein coding sequence and cloning into the Ndel site of the T7 pet expressIon vector (Studier and Moffat, 1986). The induced bacterial culture was centrifuged, and cell pellets were resuspended in Z-buffer (Desplan et al., 1985) and sonicated. The resulting supernatant was used as crude extract. Control extract was made in the same manner with bacteria carrying the expression vector without any Kr or kni coding sequences. All binding assays were performed with about 50 ng of labeled input DNA and 2.5 pg of cold salmon sperm competitor DNA. It should be noted that the actual amount of competitor DNA is much greater, since a large amount of bacterial DNA is still present in our crude protein extracts (data not shown). Five microliters of either Kr or kni antiserum was used in all reactions. Acknowledgments We thank the members of our lab for helpful suggestions on the manuscript and Wolfgang Lukowitz for discussion and Maximilian Busch for preparing the kni protein extract. We are grateful to Martin Htilskamp and Diethard Tautz for making available to us the h whole-mount in situ hybridizations and C. Niisslein-Volhard for various mutant fly stocks. The h cDNA was kindly provided by Phil Ingham. We especially thank David Ish-Horowitz for the h upstream DNA, restriction maps, and sequence information prior to publication. This work was supported by the Deutsche Forschungsgemeinschaft (Leibniz Programm) and the Genzentrum Mijnchen. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received December

15, 1989.

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