Making stripes in the Drosophila embryo

EVIEWS 14 15 16 17 18 19 20 21 22 23 24

26 27

Kelsoe, J.R. et al. (1989) Nature 342, 238-243 Bassett, A. et al. (1988) Lancet i, 799-800 Sherrington, R. et aL (1988) NaW.re 336, 164-167 Kennedy, J. et al. (1988) Nature 336, 167-170 St Clair, D. et al. (1989) Nature 339, 305-309 Detera-Wadeligh, S.D. et al. (1989) Nature 340, 391-393 Crow, T.J. (1988) Br.J. Psycbiat. 153, 675--683 Collinge,J. et aL (1989) Cytogenet. Cell Genet. 51,978 (Abstract) Elston, R.C. (1986) in Modern Statistical Methods in Chronic Disease Epidemiology, (Moolgavkar, S.H. and Prentice, R.L., eds), pp. 62-69, John Wiley & Sons Risch, N. (1984) Am..[. Hum. Genet. 36, 363-386 Clerget-Darpoux, F., Bonaiti-PeUie,C. and Hochez, J. (1986) Biometrics 42, 393-399 Donis-Keller, H. et al. (1987) Cell 51,319-337 Bodmer, W.E (1986) ColdSpring HarborSymp. Quant. Biol. 51, 1-13 Cavalli-Sforza,L.L. and King, M.C. (1986) Am.J. Hum. Genet. 38, 599--616

E d Lewis discussed over 25 years ago the concept of genetic regulatory mechanisms that control developmental pathways in Drosophila t. About ten years ago, NOsslein-Volhard, Wieschaus and JOrgens carried out a systematic search for mutations in the Drosophila genome that would affect the segmental body pattern of the developing embryo z-5. On the basis of the phenotypes of the mutant embryos, the various genes that were identified were grouped into four categories the now well-known coordinate, gap, pair rule and segment polarity gene classes, representing the different 'levels of spatial organization'. The main conclusions from the mutational analysis, laying down the basis for the concept of segmentation hierarchy, were summarized as follow#: -

'The coordinate mutants support the idea of a monotonic gradient defining position along the entire anteroposterior egg axis. The mutant phenotypes in the further three classes suggest that the subdivision of this continuous gradient into discrete positions involves unique and repetitive subfields. The single-segment repeat unit appears to be preceded by a double segmental unit.' The veracity of these arguments has been borne out by subsequent series of molecular and genetic experiments (reviewed in Refs 7, 8). One particular aspect, however, has attracted a considerable amount of both mental and experimental attention. Although it is not difficult to imagine how one continuous pattern is derived from another, or how one periodic pattern is derived from another, it is not at all clear how a periodic pattern is generated from a series of continuous ones. This problem became defined in molecular terms when the various genetically identified segmentation genes were cloned and their products visualized in the embryo. Although the mutants were isolated on the basis of the segmental phenotypes of the larva that emerges at the end of embryogenesis, the genes responsible for establishing these body patterns were expressed very early in development, before any "FIG SEPTEMBER ©1990 Elsevier Science Publishers Lid (UK) 0168 - 9479/90/$02.00

28 Martinez, M.M. and Goldin, L.R. (1989) Am.J. Hum. Genet. 44, 552-559 29 Lander, E.S. and Botstein, D. (1986) Proc. NatlAcad. Sci. USA 83, 7353-7357 30 Martinez, M.M. and Goldin L.R. Genet. Epidemiol. (in press) 31 Saiki, R.K. et al. (1988) Science 239, 487-491 32 Myers, R.M., Sheffield, V.C. and Cox, D.R. (1989) in PCR Technology. Principles and Applications for DNA Amplification (Erlich, H.A., ed.), Stockton Press 33 Weber,J. and May, P.E. (1989) Am.J. Hum. Genet. 44, 388-396 34 Risch, N. (1990) Genet. Epidemiol. 7, 3-16 35 White, R. and Lalouel,J-M. (1988) Sci. Am. 258, 40--48 E.£ G~zsuoN, M. MaRnNE~ L.R. GOL~INANDP.V. G~IMaN ARE IN THE CHNiCAL NEUROGENmCSBRANCH, NATIONAL INSI~TtrfEOFMF.UT~LH~iLr~ 10/3N21~ 9000 R~A'VTLLE Plg~ BBTHESDA,MD 20892, USA.

Making stripes in the Drosophilaembryo m ~

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The striped pattem of exprevsion of gbe Drosophila primary pair rule geaev is cot~rol~d by independent regulatory units that give rise to individual stripes. The dtfferemt s ~ p e s seem to respond in a co~etffratioudepemfe~ mamwr to the different combinations of maternal and gap protein gradiemts found along the a~erior-posterior axis of the early embrytx Thus, the initial petffodZaty appears to be generated by putting together a series of nomperiodic events.

morphological signs of segmentation. Moreover, the distribution of the gene products matched their presumptive functions: the product of the coordinate gene bicoid formed a concentration gradient along the length of the embryo, the different gap genes were found in large discrete blocks, and the pair rule and segment polarity genes were expressed in repeating stripes (Fig. 1). The regulatory hierarchy that was inferred from the genetic data was supported by subsequent molecular analyses, which showed that the expression pattern of the coordinate genes was independent of the activities of the genes in the other classes, while the gap gene expr~.ssion patterns were dependent on the coordinate genes but not on the pair rule genes. Expression of the pair rule genes, in turn, was dependent on the coordinate and the gap genes but not on the segment polarity genes. Therefore, the successive and hierarchical subdivision of the embryo was manifest in a series of molecular prepatterns that preceded any overt morphological patterning. As the bridge between continuity and periodicity involved gap genes on one side and the pair rule genes on the other, the question 1990 VOL.6 ~'~0.9

~EVIEWS raised above was posed in more cono'ete terms: how do the gap genes control the pair rule genes? In this short review, we address a very specific problem within this context and focus on the formal mechanisms through which the initial striped pattern might be established. We do not aim at completeness, but rather would merely like to discuss some recent findings that we hope will remove some of the mystery surrounding the question of how periodicity is generated in the embryo. A more comprehensive review on the control of the pair rule genes has recently been publishedg.

Gap proteins: spatial cues in the form of overlapping concentration gradients The different gap genes affect the development of large, ovedapping regions along the longitudinal axis of the embryo (see Fig. 2 for details; reviewed in Ref. 10). All the molecularly characterized gap gene products possess DNA-binding motifslH3, suggesting that they act by controlling the expression of other genes. Antibody staining revealed that the gap proteins are found approximately in the regions of the eady embryo that are affected by the gap mutations. The staining intensity within a given gap domain is not

:,

hb

uniform, but shows a graded distribution. For example, the Kr mutation affects the first thoracic through the fifth abdominal segments. However, strong staining with antibody to the Kriippel protein is observed only in the primordia of the second thoracic to the second abdominal segments, while only very weak staining can be seen in the other affected segment primordia ~4. A similar situation has been observed for the gap genes bb and bni. These results have recently led to the view that gap gene products may form steep concentration gradients within their respective domains of actionl~-17. At the same time, these data raised the thorny question of the magnitude and extent of such gradients, and whether such low, barely detectable amounts of the gap proteins are biologically relevant, or are merely visual artifacts. Recent studies on the interactions among the gap genes support the idea that such low levels of the gap proteins may indeed be functional: it appears that low amounts of hunchback and KrOppel proteins are sufficient to activate or enhance the expression of the neighboring gap genes Kr and kni, respectively17.18. Regardless of the exact mechanism by which these gradients might be established (differential gene activity in different spatial domains, diffusion of protein products, etc.), one of the conclusions from these findings was that overlapping series of gap protein gradients are formed along the anterior-posterior axis of the early embryo. The positional information provided by these gradients appears to direct the expression of the downstream pair rule genes. The next problem was to ask how the regulatory regions of the pair rule genes were organized such that they could interpret these spatial signals.

Pair rule regulation: independent control units for

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h FIGH Spatial distributions of some of the gap and pair rule gene products. The protein patterns are ~hown for bb, Kr and kni products, while the RNApattern is shown for b.

individual stripes The problem of pair rule stripe formation was initially somewhat misleading because of the similarity in expression patterns of the various pair rule genes that later proved to possess fundamentally different regulatory mechanisms. Apart from a few minor differences, all the pair rule gene products show the same basic outward appearance of seven evenly spaced stripes. When the regulatory region of one of these genes, fusbi tarazu (flz), was first analysed in detail by Hiromi et a139, it was found that a small upstream fragment of f l z fused to a reporter gene directed expression of all the stripes, suggesting that the seven stripes are controlled as a single unit. However, the work of Howard et al. 2o suggested that this may not hold true for all the pair rule genes. The)' studied mutations in the pair rule gene hairy (b) that deleted the expression of specific stripes in the early embryo. Mapping data showed that the mutations occurred in the regulatory region of the b gene, suggesting that specific elements give rise to particular stripes. Studies on the regulatory region of another pair rule gene, even-skfpped (eve), showed a similar effect: fragments derived from the eve upstream region could drive expression of a reporter gene in regions corresponding to particular eve stripes21,22. An analogous situation has been confirmed for b

TIG SEPTEMBER 1990 VOL.6

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BEVIEWS

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FIGM Schematic diagram of the expression patterns and/or domains of action of the various maternal, gap and pair rule genes. The shapes of the curves are approximate and are based on the product distribution of the genes and/or inferred from genetic studies. The etabryo at the top is divided to show the three maternal systems that establish the anterior-posterior polarity33. Zygotic hunchback (hb) mutations cause pattern deletions in gnathal and thoracic segments as well as defects in the extreme posterior abdominal segments; Kr@pel (Kr) mutations delete thoracic and anterior abdominal segments; knirps (kin) mutations affect the development of all but the last abdominal segment, while giant (gt) mutations affect parts of the head and thorax, and the posterior abdominal segments. In the terminal regions, tailless(tll) and hudebein (hkb) activities are required for the development of areas posterior to the seventh abdominal segment, as well as the e._~remeanterior parts of the embryo (for further irffc:mation see Refs 10, 34). For the posterior expression domains of gt and hb, the posterior border retracts anteriorly during development (later expression borders shown in dotted lines), bieoid (bed) is provided nmtemally and is also shown in dotted lines. The bottom portion shows the approximate positions of the hairy (is) and even-skipped (eve) stripes. There is a rely weak expression of an eighth h stripe during late blastoderm. T, terminal; A, anterior; P, posterior; proct, proctodeum; md, mandibular segment; mx, maxillary segment; la, labial segment. (Ref. 23; K. Howard, pers. commun.; G. Riddihough, M. LardeUi and D. Ish-Horowicz, pers. commun.). These results strongly supported the view that h and eve are not responding to a preset periodic pattern, but rather that they are responding to unique spatial cues that have been established along the axis of the embryo. It is noteworthy that, in general, proper f l z expression requires the activities of eve and h, whereas neither h nor eve requires f l z activity, indicating that both eve and h lie upstream of f l z in tenm of

regulatory control. Thus a regulatory hierarchy exists even among the members of the pair rule genes, with the result tb.,t '~ and eve are classified as primary pair rule genes, and f l z as a secondary onea,24,25. The conclusion from all this work was that the seemingly identical periodic patterns probably come about through two distinct mechanisms. The first one, as exemplified by h and eve, puts together the seven stripes piece by piece, following directions from a variety of nonperiodic cues. On the other hand,

TIG SEPTEMBER1990 VOL.6 NO. 9 2~ ~,

BEVIEWS f l z receives cues from an already periodic pattern to

form the seven stripes in unison. Thus, the original question can be rephrased from asking how the pair rule genes are controlled by the gap genes, to asking how each stripe of a primary pair rule gene is generated in a particular spatial location of the embryo.

(a)

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Activation and repression of pair rule stripes by the gap genes A simple, first-step approach to questions of this type has been to monitor the changes in the expression patterns of a target gene in embryos mutant for an effector gene, that is, to look at the expression

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Control of the sixth b stripe by Kr and knL (a) Wild-type situation: knirps can activate transcription only at high concentrations, while Kriippei can repress it even at low concentrations. Repression overrides activation, so that stripe expression is observed only in an area with high enough knirps concentration to activate, but not enough Kr(ippel to repress transcription. (b) kni mutant: in the absence of the knf gene product, no activation occurs. Therefore, there is no stripe expression. (c) Kr mutant: in the absence of the Kr gene product, no repression occurs. Therefore, stripe expression will be observed in the entire area of high knirps concentration. Note that knirps expression is itself decreased in Kr mutants 17. (d) Ectopic Kr expression: if Kdippel protein is expressed throughout the embryo, via heat shock control, repression will take place through the entire kni domain, and no stripe will be formed. T[G SEFrEMBER1990 VOL.6 NO. 9

EVIEWS of the pair rule genes in various gap mutant embryos and see how a particular stripe is altered. Unfortunately, interpretation of these experiments was hindered for an al:'parently trivial reason. When the expression pattern of a pair rule gene is monitored in gap mutant embryos, it is not clear how a particular stripe is affected because there is interference from the other stripes (what is usually observed in these situations is a broad continuous band of expression replacing particular sets of stripes). But with the finding that the different stripes of h and eve are generated by distinct regulatory elements that could be separated from each other, it became possible to study a particular stripe individually (or more precisely, reporter gene expression corresponding to specific stripes). Thus, for example, when the expression patterns of certain eve stripes were studied, it was found that stripe 2 expands anteriorly in gt mutants21. For h, particular stripes also expanded and disappeared23; for example, in Kr mutants stripe 5 disappears while stripe 6 expands anteriorly. In kni mutants, on the other hand, stripe 6 disappears while stripe 7 expands anteriorly. These results suggested that the gap genes can activate and repress the formation of specific pair rule stripes. As all the gap proteins analysed to date possess DNA-binding motifs, and since it has been shown that hb and Kr gene products bind specifically to the regulatory elements controlling stripes 2 and 3 of eve~6, it is most likely that the gap genes act directly on b and eve. These data also implied that the formation of certain stripes required the combined activities of two or more gap genes. In the case of the second eve stripe, it was proposed, from genetic evidence 26, that hb exerts a positive effect while Kr exerts a negative effect t6. Therefore, hb could activate the expression of eve stripe 2 while Kr blocks this activation t6. In addition, it has been shown that gt represses expression of this stripeaL Therefore, eve stripe 2 could be formed by bb activating transcription in a broad domain, and 8t and Kr setting the anterior ~nd posterior borders, respectively, by acting as repressors. For b expression, the sixth stripe is activated by kni and repressed by Kr23. The other stripes of b and eve could be similarly controlled by the combination of activating and repressing effects of the different gap genes.

Pair rule response to gap gene gradients: re~llng different concentrations through binding sites of differing affinities? We now have the situation that gap proteins can activate or repress specific stripes, and that a particular pair rule stripe can be controlled by different gap genes. The sixth h stripe, for example, is regulated by both Krtippel and knirps. However, Kriippel and knirps proteins are found M broad regions of the embryo. How is it, then, that this stripe is only found in a very narrow region in the posterior part of the embryo? A potential explanation emerges from in vitro DNA-binding studies with Kriippel and knirps proteins with the different control units from the b gene that generate particular stripes z3. These data showed that Krtippel protein binds with high affinity

to h control elements that direct expression of stripes in an area of low KriJppel concentration, while binding with low affinity to control elements that generate stripes in areas of high Krtippel concentration. For example, stripes 3 and 4 lie in an area of high Krtippel concentration, while stripe 6 lies in an area of very low KriJppel concentration. In this case, Kriippel protein binds to the control element that generates stripe 6 much more strongly than to the control element that generates stripes 3 and 4. This inverse correlation between binding affinity and gap protein concentration is also observed for knirps. Stripe 6 lies in an area of high knirps protein concentration, while stripe 7 lies in an area of very low kni expression. In this case, knirps protein binds to the stripe 6 element very weakly, but binds strongly to the stripe 7 element. These results could help to explain how the expression of h stripe 6 is spatially restricted to a narrow domain. As stated above, this stripe is activated by kni and repressed by Kr. Furthermore, knirps protein binds to the control element that generates this stripe very weakly, whereas Krtippel protein binds strongly. Therefore, the b stripe 6 control element, due to the presence of low-affinity knirpsbinding sites, would be activated only by high knirps concentration; however, Krtippel would repress this element even at very low levels due to the presence of high affinity KriJppel-binding sites. If we assume that repression can override activation, this element will only be active in an area where knirps concentration is high enough to activate, but where Krtippel concentration is too low to repress transcription. Because of the particular distributions of the two gap proteins, this compromise situation would be found only in a unique position along the length of the blastoderm embryo - where stripe 6 is located. This would also explain the behavior of the sixth h stripe in different mutant embryos (Fig. 3). In the absence of an activator, as in a kni mutant, there would be no expression. Without the repmssor, as in a Kr mutant, the expression would be broadened to coincide approximately with the activator domain. Furthermore, if repression can indeed override activation, ectopic presence of Kriippei in the knirps region should block the activation of ~,tripe 6 by kni. This is in fact what is observed when Kr is expressed at low levels tPLroughout the embryo under heat shock promoter control: the expression of the sixth b stripe is suppressed (M.J. Pankra*oz, unpublished). This manner of generating a stripe is analogous to the mechanism by which bcd activates bb expression. Work by Driever and Ntisslein-Volhard 27, and by Strum et aLas, showed that bb responds to the bicoid gradient in a concentration-dependent manner through the presence of different affinity bicoid-binding sites. For the control of a stripe by the gap genes, one now needs to add an additional component - a different gradient of a repressor that overlaps the first activator gradient. The final outcome would be the result of the superimposition of the two distinct gap gene activities. The range of concentration values at which the target promoter is activated or repressed would be

TIG SEPTEMBER1990 VOL.6 NO. 9

El

[~qEVIEWS

determined by the affinity of binding sites within the control unit for the respective gap proteins constituting the gradient.

Converting continuous spatial information into periodic pattern

Acknowledgements We thank S. Cohen, J. P. Gergen, M. Hoch, G. Jiirgens, M. Klingler, M. Levine, D. Tautz and an anonymous reviewer for their very helpful suggestions on the manuscript.

References

The above studies suggest that periodicity p e r se arises by a not very elegant mechanism-'9: seven independent events added together. Therefore, the question of how a periodic pattern is generated in the Drosophila embryo could be broken down into asking how a series of individual, nonperiodic patterns arise. Of course, it is no small matter to ask then how each of these events occur. The scenario outlined above for the sixth b stripe is a minimal version with which to illustrate how two components could in principle generate a stripe. Kr and kni by themselves almost certainly do not provide enough information to generate a 'biologically complete' sixth b stripe. There are probably additional factors that help to activate, repress, enhance, or maintain the expression of this stripe, including other gap and pair rule genes. For instance, the posterior border of the sixth b stripe may be set not only by lack of activation by kni, but also by repression from tll. In addition, gt could enhance b stripe 6 expression, since the expression of this stripe seems to be decreased in gt mutant embryos (M. Busch, M.J. Pankratz and H. Jiickle, unpublished). Thus, the initial information provided by the gap genes is probably sufficient to make only a crude ~pproximation of a stripe, with low plateaus and fuzzy boundaries. Once an initial, coarse periodic pattern has evolved, interactions among the pair rule genes themselves would refine the stripes to their final form by sharpening the borders and making the peaks more distinct. What of the other stripes of b and eve? Some of these could be formed in an analogous manner to that described above for she second eve and the sixth b stripes. Others may not. Each stripe is located in a region of the embryo with a distinct set of gap gene combinations and concentrations. Furthermore, maternal factors may also have direct influence on certain pair rule stripes, for example, bcd could be involved in b stripe 1 formation30 and in eve stripe 2 activation (M. Levine, pers. commun.). It would therefore not be surprising if the different stripes used slightly different mechanisms specially suited for their own need. Even if a formal mechanism could be found for all the stripes of b and eve, a whole set of questions and complexities still remains. How can small differences in gap protein concentrations bring about a threshold response, be it activation or repression? What is the nature of the interactions that sharpen the pair rule stripes? What mcchanism is responsible for aligning the stripes of the different pair rule genes so precisely relative to each other? Due to space limitations we have not touched upon issues that will certainly have bearing on these problems, such as the interactions among the primary pair rule genes and the regulatior, of the secondary pair rule genes (discussed further in Refs 9, 30--32). Conceptually, however, these problems have more to do with pattern refinement and maintenance than with generating periodicity.

1 Lewis, E.B. (1963) Am. Zool. 3, 33-56 2 NOsslein-Volhard, C. and Wieschaus, E. (1980) Nature 287, 795--801 3 Ntisslein-Volhard, C., Wieschaus, E. and KKiding, H. (1984) Roux's Arch. Dev. Biol. 193, 267-282 4 JOrgens, G., Wieschaus, E., Niisslein-Volhard, C. and KlOding, H. (1984) Roux'sArch. Dev. Biol. 193, 283-295 5 Wieschaus, E., Ntisslein-'vblhard, C. and JOrgens, G. (1984) Roux~ Arch. Dev. Biol. 193, 296-307 6 NOsslein-Volhard,C., Wieschaus, E. and Jtirgens, G. (1982) Verh. Dtsch. Zool. Ges. 91-104 7 Akam, M. (1987) Development 101, 1-22 8 Ingham, P. (1988) Nature 335, 25--34 9 Carroll, S. (1990) Cell60, 9-16 10 Lehmann, R. (1989) Development 104 (Suppl.), 17-27 11 Rosenberg, U. et al. (1986) Nature 319, 336-339 12 Tautz, D. et al. (1987) Nature 327, 383-389 13 Nauber, U. et al. (1988) Nature 336, 489-492 14 Gaul, U. and J~ickle, H. (1987) Trends Genet. 3, 127-131 15 Gaul, U. and J~ickle, H. (1989) Development 107, 651--662 16 Stanojevic, D., Hoey, T. and Levine, M. (1989) Nature 341,331-335 17 Pankratz, M., Hoch, M., Seifert, E. and Jiickle, H. (1989) Nature 341,337-340 18 Htilskamp, M., Pfeifle, C. and Tautz, D. Nature (in press) 19 Hiromi, Y., Kuroiwa, A. and Gehring, W. (1985) Cell43, 603-613 20 Howard, K., Ingham, P. and Rushlow, C. (1988) Genes Dev. 2, 1037-1046 21 Goto, T., Macdonald, P. and Maniatis, T. (1989) Ceil57, 413--422 22 Harding, K., Hoey, T., Warrior, R. and Levine, M. (1989) EMBOJ. 8, 1205-1212 23 Pankratz, M. et al. (1990) Cell61, 309-317 24 Howard, K.and Ingham, P. (1986) Cell44, 949-957 2.5 Carroll, S. and Scott, M. (1986) Cell45, 113-126 26 Frasch, M. and Levine, M. (1987) Genes Dev. 1, 981-995 27 Driever, W., Thoma, G. and Niisslein-Volhard, C. (1989) Nature 340, 363-367 28 Struhl, G., Struhl, K. and Macdonald, P. (1989) Cell 57, 1259-1273 29 Akam, M. (1989) Nature341, 282-283 30 Hooper, K., Parkhurst, S. and Ish-Horowicz, D. (1989) Development 107, 489-504 31 Ingham, P.W. and Gergen, J.P. (1988) Development 104 (Suppl.), 51-60 32 Dearolf, C., Topoi, J. and Parker, C. (1989) Genes Dev. 3, 384-398 33 N0sslein-Volhard, C., Frohnh6fer, H.G. and Lehmann, R. (1987) Science 238, 1675-1681 34 Weigel, D., JOrgens, G., Klingler, M. and Jiickle, H. (1990) Science 248, 495--498

M~. PANlCRA?ZAND H. JACKLE ARE IN THE INSTIT~ FOR GENETIK UND MIKROBiOLOGIF~ UNIVERSITAT MONCHEN, MAma WARDSTg 1/6 8 0 0 0 MONCH~.N19, FRG.

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