Biochbnica et Biophysica Acre, 1114 (1992) 79-93

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@ 1992 Elsevier Science Publishers B.V. All rights reserved 0304-419X/92/$05.00

BBACAN 8725O

Transcriptional regulators of Drosophila embryogenesis Douglas R e a d a n d J a m e s L. M a n l e y Department of Biological Sciences, Columbia Unieersity, New York, NY (USA) (Received 5 March 1992)

Contents 1.

Inlroduclion

II.

Positional information in DIvJ,sophila emhl)tollenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The dorsal°ventral body axis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The anterior-posterior body axis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Zygotic segmentation genes . . . . . . . . . : ......................................

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

79 79 80 ~() 81

!!1. Transcriptional regulation in Drosophila embryogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. DNA binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Transcriptional regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

81 82 82

IV.

Regulati,~n of eve and ftz striped expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Reguk~tion of eve and ftz expression in trans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Reguiation of ere and .ftz expression by cis clemeuis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

83 84 85

V.

An in vitro approach to identify Drosophila transcription factors . . . . . . . . . . . . . . . . . . . . . . . .

86

VI.

The role of tramtrack in ere and fiz regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

89

VII. Summary and Perspectives References

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91

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!. Introduction

One of the fundamental issues of biology today is the question of how individual metazoan cells acquire their unique identities. Partial solutions to this problem have been achieved in the last decade, in large part through the study of embryogenesis in Drosophila melanogaster. Classical notions of morphogens as determinants of spatially restricted zygotic gene activity, and the role of differential gene expression in the determination of cell fate, have been firmly established. This review introduces the systems of regulatory interactions between genes involved in pattern formation during early stages of embryogenesis. Particular emphasis is given to interactions occurring at the level of transcription initiation, and results of biochemical

approaches aimed both to identify novel transcriptional regulators, as well as to understanding the molecular mechanisms underlying the function of genetically defined regulators, are reviewed. Regulatory interactions involved in controlling expression of the pair-rule segmentation genes even.skipped and fushi tarazu are described in some detail as exainplcs of complex transcription units in which both genetic and biochemical methods have contributed to our understanding of their regulation (see Refs. 15, 46 and l l0 for related reviews). (A number of other relevant reviews are referred to throughout the text.) The review concludes with a discussion of the possible role of the tramtrack gene, which was identified solely by biochemical ap,, proaches, in controlling the onset of ~gatic tran~cripo tion of these t~:o genes. 11. Positional information in Drosophila embryogenesis

Correspondence to: J.L. Manley, Department of Biological Sciences, Columbia University, New York, NY 10027, USA.

In Drosophila, the first 13 nuclear divisions following fertilization occur in near synchrony and in the

80

e ~ n c e of cellularization. During these initial cleavages, most of the nuclei migrate to the embryo cortex, giving rise to a monolayer of about 5000 hexagonaily packed nuclei surrounding the central yolk mass [31.132], ResuRs of nuclear transplantation experiments indicate that at this stage nuclei are not committed to a particular developmental fate. By the end of the 14th cleavage cycle however, the fate of each nucleus is determined according to its position within the embryo [56.66,114.131]. An understanding of the acquisition of cellular identity requires that we first learn how positional information is established within the embryo and, then, by what mechanisms individual cells interpret this information to follow specific developmental pathways, Considerable progress has recently been made in the effort to understand the mechanisms underlying the elaboration of positional information in early D e o ~ i l a embryogencsis. Much less is currently known about processes involved in the determination of cellular identity. A simple way to specify the location of an individual cell in an elipsoidal monolayer of cells is to identify its position along each of two axes. it should not be surprising then that distinct systems responsible for establishing positional values along the anterior-posterior and do~i-ventral axes of the Drosophila embryo have in fact been identified (reviewed in Refs. 87 and 102). The existence of graded distributions of cellular determinants in insect embryos had been inferred from experiments involving ligation, ablation, and transplantation of cytoplasm, and from analysis of mutant pheno~os, Such 'morphogens' wcrc beiieved to be deposited during oogenesis. Recent molecular and goattic approaches in Droso~tila have provided direct evidence for the role of maternal morphogens in the establishment of the dorsal-ventral and anterior-post. crier body axes of the Drosophila embryo (reviewed in

Refs, ~, 87 and 102), ~ e realization that the visible defects resulting tram embryonic lethal mutations could be instructive led to the identification through saturating mutagenesis screens of at least 100 genes that function in the estabibhment of the anterior-lxxsterior and dorsalventral patterns [65,74,83,84,105,106,125]. These are classified as maternal or zygotic mutations according to whether the phenotype is dependent on the genotype of the mother or on that of the fertilized embryo. The epistatic relationships among these genes provide a picture of two sets of regulatory cascades in ~,'hich ceud¢ ~ i t i o n a l information supplied during oogenesis is progressiveky refined through the actions and interactions of z~gotic genes (reviewed in Ref, 87),

II-A. The dor~l-~¢ntrai body ¢¢is AIthou~ mechanistically complex, the dorsal-ventral system is simpler than the anterior-posterior system in

the sense that it consists of a single system of interact~ng gene products that functions to establish positionspecific cues to which zygotic genes respond (for reviews, see Refs. 2, 72 and 100). Recessive alleles of 11 maternal-effect genes have been identified, all of which give rise to dorsalized embryos, i.e,, embryos in which structures associated with ventral regions are replaced by more do~al structures [82,106]. Epistatic relationships among these dorsal group genes have been deduced by genetic means and by cytoplasmic transplantation experiments, The principal conclusion of such studies is that the product of the dorsal (dl) gene is the primary determinant of positional information along the dorsal-ventral axis. A number of dl alleles can be arranged in a series of progressively more severe phenotypes, suggesting that in wild-type embryos there is a graded distribution of dl function. Direct visualization of the dl protein within embryos revealed that while the protein itself is not distributed in a simple concentration gradient, its nucleo-cytoplasmic distribution varies along the dorsal-ventral axis [99,101,113]. The dl protein contains sequences homologous to the DNA binding and aimerization domains of the pS0 and p65 subunits of NF-•B, a mammalian transcription factor whose activity is regulated at the level of nuclear localization, and considerable experimental evidence indicates that dl regulates the expression of zygotic genes at the level of transcription [21,57,101,119]. Binding sites tbr the dl protein have been shown to be required for the all-de. pendent regulation of the ~,gotic genes zerkniillt and twist [21,57,62a,89]. Furthermore, subtle changes in the dl nucleo-cytoplasmic gradient are observed in embryos mutant for certain of the dorsal group genes, including dl itself, and these changes in the gradient are faithfully reflected in altered patterns of zygotic gene expression [99]. So, while dl does not fit the classical notion of a morphogen as formulated in terms of a protein concentration gradient, with respect to the distribution of its function, dl clearly has the essential properties of a morphogen.

ll.B. The anterior-posterior body axis in contrast to the dorsal-ventral system, the determinants of positional information along the anteriorposterior axis constitute three discrete systems of genes, each responsible for the placement of spatial cues within a particular region along the axis [86,87,102]. The anterior and posterior group genes function within the segmented regions of the head and thorax, and of the abdomen, respectively. The terminal class of genes is required for differentiation of structures in the unsegmented regions at the extreme poles of the embryo. The function of the anterior group gene bicoid (bcd) has been especially well characterized. During oogene-

81 sis, bcd RNA is localized to the anterior pole of the oocyte. Mutations in the maternal-effect genes exuperantia and swallow disrupt bcd RNA localization, resulting in its distribution throughout the oocyte [36]. Diffusion of the bcd protein following its translation in the fertilized zygote results in a higher local concentration of the protein in the anterior end than at the posterior end of the embryo [25]. The shape of this concentration gradient can be manipulated by varying the number of copies of bcd in the genome, and increases and decreases in gene dosage result in the predicted posterior and anterior shifts in the blastoderm fate map. The altered fate map is reflected in the larval cuticle and in corresponding shifts in the domains of expression of zygotic genes [26]. The bcd protein contains a horace-domain that mediates binding to regulatory regions of zygotic segmentation genes and like dl, bed has been shown to function as a transcriptional regulator [23,111,115,117]. The response of target promoters to the bed gradient can be artificially manipulated by varying the number and relative affinity of bed protein binding sites that the promoter contains, providing further evidence that zygotic genes respond to positional values along the bcd gradient [23,24,111]. The bed protein thus fits the classical definition of a morphogen.

to analyse the flow of intormation from the maternaleffect genes through the zygotic segmentation genes by directly visualizing expression in mutant embryos (reviewed in Refs. 1 and 56a). The three systems of anterior-posterior maternal-effect genes function to define the limits of gap genc expression, and mutually repressive interactions between gap genes are also important for defining these limits. In turn, the borders and overlapping domains of gap gene expression determine the spatially restricted expression of the pair-rule genes. Interactions between members of the pair-rule class further refine the resulting striped patterns of expression, and pair-rule genes can be classified as primary or secondary according to whether or not they can exert such an influence on other pair-rule genes. Specific combinations of pair-rule gene products occurring within cells of each segment-length domain determine the expression of the segment-polarity genes. In response to positional cues laid down by the segmentation genes, the homeotic genes function later in emo bryogenesis ~,o determine the identity of particular segments. As discussed below, most of the regulatory interactions within this hierarchical system occur at the level of transcription.

IlL Transcriptional regulation in Drosophila embryogenesis

H~C. Zygotic segmentation genes In addition to the systems of maternal-effect genes, mutagenesis screens have led to the identification of many zygotic genes whose mutant phcnotypes indicate a role in establishing spa'.~! domains within the two embryo axes [65,83,84,125]. The zygotic genes that contribute to the formation of the segmentally repeated pattern of structures along the embryo anterior-posterior axis are collectively referred to as segmentation genes [83, reviewed in Refs. 1 and 56a). These can be classified according to the extent of the pattern that is disrupted in the terminally differentiated phenotypes of mutants. Embryos mutant for any one of the five known gap genes lack structures associated with contiguous blocks of segments, and structures in the remaining segments are unaffected. Mutations in pairrule genes give rise to phenotypes in which pattern elements of alternate segments are absent. Finally, in embryos mutant for segment polarity genes, portions of each segment are missing, often being replaced by mirror-image duplications of the remaining portion of the segment. Thus, the gap genes have broad functional domains that include multiple segments, while the pair-rule genes define alternate segment-length domains, and the segment-polarity genes specify domains within each segment. Once molecular probes for products of many of the segmentation genes became available, it was possible

The first homeotic genes were cloned by conventional techniques based on genetic mapping data. Within the coding sequence of the Antennapedia gene was found a 180 bp sequence common to a number homeotic genes and to the pair-rule gene fushi tarazu (ftz) [76,!09]. The homology of the encoded homeo domain to sequences within known transcriptional regulators in bacteria and yeast provided the first indication that the segmentation and homeotic genes might themselves function by controlling the transcriptional activity of other genes [70,76]. This idea was supported by the finding that a small portion of the engrailed (en) protein that contained the homeo domain could in fact bind DNA in a sequence-specific manner [19]. While the term homeobox originally referred to sequences sharing a high degree of sequence identity (80-90%), this stringent use of the term has been relaxed as a large number of less similar genes have been identified in a wide variety of organisms. The use of the homeobox as a hybridization probe allowed the rapid isolation of many of the pattern forming genes in Drosophila. Over half of the more than 30 Drosophila homeobox genes isolated to date are either segmentation or homeotic genes that had been previously identified by genetic means [73]. Cloning of those pattern forming genes that lack a homeobox has depended on the more difficult and time consuming methods of chromosome walking or by

P~lement mutagenesis/tagging. Many of these, however, have been shown to contain other known DNA binding sequences, including zinc finger, leucine zipper and helix-loop-helix motifs. The availability of molecular probes made it possible to examine directly the patterns of expression of these genes. In general, the pattern of expression of a given gone is consistent with the pattern of defects observed in embryos mutant for that gone. For exampie, embryos homozygous for mutant alleles of the gap gone knirps (kni), which in the cellular blastoderm stage is expressed in a broad domain corresponding to the presumptive abdomen, give rise to larvae retaining only a small portion of the abdominal region. The terminal and thoracic regions of kni ° embryos are essentially normal, The correspondence of domains of expression and function is oven more striking for the ~irorul¢ and segment polarity genes, with their more finely resolved stripes of expression and proporuonatcly narrower bands of disrupted tissue in mutant phenolypes, Studies in which the pattern of expression of a particular gone was examined in various mutant backgrounds led to a description of the segmentation proce~ in terms of a hierarchy of gone interactions. These intQractions result in the progressive subdivision of the embryo into homologous segments and segmental compartments which later acquire unique identities. Considerations of timing and the gone,rally close correspondence of the patterns of mRNA and protein expression were ~:onsistent with the idea that the protein products of the segmentation genes functioned as transcriptional regulators of subordinant genes in the hierarchy.

Ill+A. DNA bimt&g Additional support for the idea that many of the interactions among the segmentation and homeotic genes occur at the level of transcription came from studies showing that the borneo box in fact constitutes a DNA binding domain (e.g., Refs. 4, 19, 20, 52 and 53). These studies also identified binding sites for horace-domains within regulatory sequences of some g~nes Using immunoprccipitation a.~ays, Ref. 52 iden. tiffed multiple binding sites for bacterially synthesized ere protein upst~am of the engrailed fen) and ecensk/p/~d (eta,) genes. The presence of these sites was consistent with previous results indicating a role for e~'e in the regulation of e~+, and with other data suggesting that et~ regulates its own expression. Surprisingly, however, the sequences bound in the en gone do not resemble t ~ in the e+~ gone; while the en sites a ~ A-T rich, the eve sites are G-C rich. Simple notions of each horace-domain having a unique binding speci~ were further eroded by the findings that the zeeknFdt (ten), paired ( prd), and en proteins also bound

the sites in the etz gone, and that the zen and o+ proteins only had a very weak affinity tbr the eve sites. Binding experiments in which homeoboxes were mutated, or were exchanged between proteins, indicated that sequences both within and surrounding the horace-domain influence the selectivity and relative binding affinity of horace+domain proteins [20,53]. While firmly establishing the DNA binding activity of the horace-domain, these studies provided support for two models for the regulation of transcription by homeo-domain proteins which, when stated in their extreme forms, are exclusive [52]. The observed differences in the relative affinities of the horace-domain proteins for a single binding site are consistent with a model in which the on/off state of a promoter is determined through competition among horace-domain proteins for binding to sites within regulatory elements. The highly selective binding of some proteins to distinct classes of sites supports a model in which combinations of homeo-domain proteins bound to qualitatively diverse sites determine the activity of a promoter, it was concluded that it is likely that both mechanisms operate, in rive [52]. As discussed below, considerable evidence for both mechanisms has accumulated through the functional dissection of regulatory elements.

III-R Transo+iptional teen,darien The first direct evidence that homeobox genes encode transcriptional regulators was provided by transient transfection assays using Drosophila cells in tissue culture [24,41,60,68,115,126,133]. In these experiments Schneider L2 cells, which do not appear to express any of these developmental regulators endogenously, are cotransf¢cted with p!asmids that constitutively express high levels of the putative regulator, along with reporter constructs consisting of the regulatory elements of potential target genes fused to the coding sequence of the easily assayable E. coil chloramphenicol acetyl transferase (CAT) gene. Such assays demonstrated Lhat the fragment of the en gone containing multiple horace-domain binding sites can in fact function as the regulatory target of a number of homcobox genes [41]. Specifically, it was found that multiple copies of this element mediated up to 400-fold activation by the horace-domain products of the z2, ten, paired (prd), and ftz genes. More strikingly, even higher levels of activation were observed when combinations of plasmids expressing different homeobox genes were introduced along with the reporter construct. The combination of ten, ftz, and prd expression vectors resulted in synergistic activation that was more than 125-fold greater than the activation that would be expected if the combination functioned additively. To explain these results, a model was proposed

83 in which combinations of proteins bound to adjacent sites enhance transcription either by forming a more stable complex or by making more, or stronger, contacts with the basic transcription complex. It was also found that the en and eve gene products, aside from having no activating effect, were able to repress the activation by individual homeo-domain proteins, and by synergistic combinations [41]. Cotransfection assays were also used to show that activation by the ftz protein can be antagonized by the eve and en proteins [60,61], and that the repression mediated by the en protein is not due simply to competitive binding, but involves a discrete functional domain that can function when fused to a heterologous DNA binding domain [61]. Similar studies showed that the zinc finger containing products of the gap genes hunchback (hb) and griO~pel (gr) and the homeobox containing homeotic genes UItrabithorax ( Ubx ) and Antem~apedia ( Antp ) also function as transcription factors. The gap gene hb encodes a zinc finger protein that was shown to have the intriguing property of functioning as an activator at moderate concentrations, but as a repressor at both low and very high concentrations [133]. In addition to its role as a sequence-specific repressor [75], the Kr protein also has the ability to block activation by the hb protein in the absence of a Kr binding site [133] and to function as an activator at low concentrations [103]. Although the mechanism(s) underlying these concentration-dependent differences in activity is (are) unknown, tl,is property may play an important role in the function of these gap proteins in embryogenesis (see below). Both the Ubx and Antp proteins were shown to have positive autoregulatory functions in transfection assays, but wlfile the Ubx protein represses Antp '~ranseription [68], the Antp protein activates Ubx [126]. These functions were dependent on sequences downst~'eam of the respective start sites that are capable of binding both proteins [68,126]. As discussed below, cotran~fection has also been used to support a model for the generation of a single eve stripe through activation by the bcd and hb proteins, and repression by giant (gt) and Kr proteins [115]. An important question regarding cell culture experiments is whether they can be used to predict regulatory interactions that occur in the embryo. That is, does the fact that a particular protein affects the expression of a specific target gene in a transfection assay mean that the same interaction occurs in the embryo? Indeed, several examples where this appears not to be the case have been documented (see Ref. 46 for review) and, as a result, the value of the cotransfection assay has been questioned. However, this view seem:, unjustified, as it both fails to acknowledge certain limitations of a strictly genetic approach to the definition of regulatory pathways, and also does not appreciate the principal values

of transfection assays. An obvious difficulty with man~ genetic analyses is determining whether an effect is direct or indirect. That is, does gene a directly regulate gene c, or does it. act through an intermediate, gene b? A more subtle concern has to do with the issue of redundancy (see below for discussion of a possible example). Thus, when mutation of a possible regulatory gene (gene a) has no detectable effects on expression of a putative target (gene b), is this because gene a does not play a role in regulation of gene b, or because in the absence of gene a, another gene, gene c, is able to perform the same function? Thus, while it is true that results obtained in transfection experiments do not necessarily always reflect regulatory interactions that occur in the embryo (e.g., a protein-DNA interaction that occurs in cultured cells may be 'over-ridden' in embryos by other proteins that may not bc present in the cultured cells), it is perhaps worthw|lile to consider all types of analyses when trying to define precise regulatory pathways. Transfection assays (as well as in vitro transcription experiments; see below) do provide important information regardless of their ability to predict regulatory pathways in the embryo. For example, as described above, such assays can indicate whether a specific protein can function as an activator or a repressor. What are the effects of varying the protein concentration, and how do specific activators work in combinations? With respect to the latter, the demonstration that a combination of homeobox proteins, with appazently identical DNA binding specificities, can activate transcription of a target gene much more effectively than can a similar concentration of only one of the proteins t. fers a molecular mechanisin that may help explain the idea that it is the combination of activators present in a particular cell in the embryo that determines its fate.

IV. Regulation of eve and flz striped expression The first descriptions o f the patterns of eve and ftz expression were performed using radiolabeled antisense DNA probes hybridized in situ to sections of fixed embryos [40,42,77]. Both transcripts were initially detected at low levels throughout the embryo following the ninth cleavage. It is possible that transcription is activated during the previous cleavage stage, but that transcripts have not accumulated to sufficient levels to allow detection with radiolabeled DNA probes. In any case, after the tenth nuclear cleavage, eve and flz transcripts have accumulated in the middle region of the embryo, having disappeared at the poles. By the completion of tile twelfth cleavage, regional differences in the accumulation of transcripts are observed, and early in the next cycle broad bands of expression appear. Over the course of the last syncitial cleavage

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[14,33]. The patterns of ere and .fi'z expression are altered in mutants for each of the five gap genes and a subset of the pair-rule genes. Mutations in none of the segment polarity genes tested have an effect on either ere or ftz expression. Evidence that the gap genes function to establish the initial patterns of pair-rule gene expression, while the pair-rule genes serve to maintain and refine the patterns, implies a temporal component of the functional hierarchy [33]. A stady in which the patterns of expression were analysed in embryos simultaneously stained for both ere and ft: proteins revealed that both are regulated in a complementary fashion by the same set of genes [33]. For example, in kni- embryos, ece stripes 4, 5, and 6 are absent during early gastrulation, and the corresponding region is occupied by a single broad band of ft: expressing cells. In addition, while all but one of the ece stripes are abnormally broad in nmt embryos, the f~z stripes narrow prematurely and are weaker overall. In hahy- (h) embryos, the converse effects on ere ant~ j~z expression were observed [33], and ectopic expression of h has been shown to abolish

stage, the seven-stripe patterns of ere and ftz expression evolve, such that by the completion of ccllularization, each stripe encompasses about three or four cells and is separated from adjacent stripes by three to tive cells. By this time, the patterns of e~'e and ftz expression are precisely complementary. The striped patterns persist through gastrulation and early germband extension, and during this time seven additional weak stripes of ece expression appear. Late in the germband extension stage, both striped patterns gradually disappear, and during the later half of embryogenesis, both are expres~d in subsets of cells in the central nervous ffstem. The evolution of the patterns of ere and ftz protein accumulation, although delayed, parallel the accumulation of transcripts [13,32].

IV.A, Regulation of ere and ftz expressio, i~t trans Analyses of the expre.~sion of et'e and ,ftz in embryos mutant for other segmentation genes support the idea of a functional hierarchy that operates to subdivide the embryo into progressively smaller domains

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Fig, h Summary el" identified regulatory elements in the (*~(,and ftc genes and k~ations of binding sites for the 69 kDa ttk protein. Sequences u~am o~' the tr~n.,~dption stuart sites of the ~,¢ and ft: genes are resptmsible for early expression in striped patterns and for later ~ i n t e n a ~ ' e of stri~d exp~x,~h~n tha~ugh tmsitivc autoregulation, Binding sites h~r the ¢~9 kDa ttk protein arc indicated by hatched ovals. I S k ~ n t s ~'qui~d t~r generation o{ o v stripes 2 {rod 7 and stripe 3 have been identified, Sequences necessary filr the generation of stripes 2 and T h a ~ not )~! ~ c n ~ptlr~ted, pl~babl~, I~¢ause they overlup or shar~ factor binding sites. Expression of ere stripe I is weak in construcls ~,'~t~ttic~g 8 kb o~"upstream ~;quen~'e, but stripes 4, $ and f~ presumably lie further upstream. A minimal autoregulatory sequence (MAS) of 200 bl~ k ~ a i ~ ~thin the 0,? kb t~uto~gulatory elemen~ (AR) is sufficient for autoregulat;on by the err, protein and contains multiple binding sites the ¢~e pco~ein and a sidle binding sit~ for the 69 kDa tt£ protein, Late striped expression in constructs lacking the region between - 39 hp a~l -0,4 kb is weak ;~nd variable. The 0,7 kb zebr,~ element is sufficient for expression of all 7 [tz stripes and contains two binding sites for the 69 kD,~ ttk p¢o~i~. The Its. upstream element (USE) is composed of multiple elements that mediate .(tz autoregulation in a tissue-specific ~ ¢ ~ ¢ r . One bindin~ site for the 69 kDa ttk protein has been located between the proximal (P) and distal (D) elements of the USE. Not deleted in the figure is the [ ~ neurogenic region, which is located downqream of - 2.45 kb. Also not shown are the GAGA-factor binding sites, which h a ~ been identified within the ere promoter region and autoregulatory element and within the J'tz zebra element; consensus recognition sequences for the GAGA-factor occur at multiple site~ ~,ithin the ftz autoregulatory element.

85

ftz expression [58]. However, while the ftz pattern is altered in eve~ embryos [34,42], the eve pattern of expression is not affected by the absence of wild-type ftz activity. Considerable evidence also exists to indicate that the eve and ftz proteins both possess positive autoregulatory func6ons. Mutations within the eve coding sequence lead to abnormal spacing of the eve stripes in gastrulating embryos. In these mutants the striped pattern of eve expression decays prematurely during germband extension [34]. An upstream element that contains multiple eve protein binding sites has been shown to be sufficient for eve autoreguln tit, n ([39,43,62], see Fig. 1). The mechanism by which !he eve protein mediates positive autoregulation may be dependent on ancillary factors or on the particular comext of the binding sites within the autoregulatory element, since in cotransfection and in vitro assays the ere protein has as yet only been shown to function as a represser [7,41,60]. Autoregulation by l'tz has also been demonstratod through ectopic ftz expression [59], and an element mediating this activity has been identified ([51,95], see Fig. 1). II/-B. Regulation of eve and ftz expression by cis elema~ts The almost precisely complementary patterns of expression of the ere and .ftz genes, along with their dependence on the same set of trans-regulating factors, might lead one to suspect that their cis-regulatory elements are organized in similar ways. This is clearly not the case, as numerous studies involving P-element transformation with recombinant promoters driving expression of the lacZ gene have revealed [12,17,18,39, 43,50,51,62,95,111,115,123]. Dissection of the ftz regulatory region indicates that it can be divided into three functional domains that '~i~an approx. 6 kb: a 0.7 kb 'zebra' element that is sufficient for directing striped expressic ~, a neurogeni¢ domain that lies upstream of the zebra element, and an additional upstream element that has properties of an enhancer ([17,50,51,95], see Fig. 1). in contrast to the compact ftz zebra element, sequences required for the striped pattern of eve expression are distributed throughout a region spanning at least 8 kb upstream of the transcription start site, and discrete elements are responsible for mediating the expression of individual stripes, or pairs of stripes ([39,43,115], see Fig. 1). Discrete stripe-generating elements have also been identified in the h gene [55,90,98]. While a neurogenic element has not yet been identified in the eve gene, an element with enhancer properties exists approx. 5 kb upstream of the transcription start site [39,43,62]. The function of the ftz zebra element has been systematically analyzed using embryos transformed with mutagenized promoters fused to the lacZ reporter

gene [12,17,122,123]. The principal conclusion of these studies is that the striped pattern of ftz expression is generated through general activation of transcription throughout the germband, and localized repression in tile ftz interstripe regions. A similar conclusion had previously been drawn from studies in which embryos were injected with inhibitors of transcription and protein synthesis [28]. Numerous binding sites for nuclear proteins exist in the zebra element, and ~t has been shown that selective removal of particular sites can cause either general loss of lacZ expression or the derepression of expression in the interstripes [12,17,123]. Sites have also been identified that appear to have a dual function, as their removal results in low levels of expression along the length of the germband [17]. Oligonucleotides containing these three types of binding site fused to basal promoters retain the functions iJLdicated by the deletion studies [122]. Consistent with the results of analysis of ftz expression in mutant backgrounds, the negative effect of one these sites wa.~ dependent on wild-type h functioiJ [122]. In addition, a functional caudal protein binding site has been identified in the zebra element [18]. The initial studies that identified the enhancer properties of the upstream element also showed that its function is dependent on wild-type ftz activity [51]. Evidence was also provided for tissue-specific regulation of ftz expression, for while the zebra element directed lacZ expression that is predominantly meso. dermal, the upstream element is capable ot mediating expression in both the mesoderm and the ectoderm [51]. A systematic dissection analysis of the upstreatn element led to the identification of two discrete components with enhancer properties: a distal element that directs expression in the mesoderm, and a proximal element that directs expression in both the mesoderm and the ectoderm [95]. The proximal element itself is composed of two sub-elements, one of which is capable of activating mesodermal expression. The other subelement, while it has no independent function, is required for expression in the ectoderm. The proximal and distal elements both contain multiple ftz protein binding sites, supporting the idea that the positive autoregulation mediated by the upstream enhancer element results from a direct interaction [95]. As was mentioned above, the organization of the elements governing striped expression of ece is quite distinct frem that of the ftz zebra element, in the respect that deletion of far upstream sequences results in the loss of individual stripes or subsets of stripes, the eve regulatory region is similar to that of h [55]. More than 8 kb of eve upstream and promoter sequence fused to the lacZ gene was sufficient to direct strong early expression only of stripes 2, 3, and 7; stripe I was barely detectable [43]. The sequences required for stripes 4, 5, and 6 have not been identified. Individual

elemenB have been identified that are responsible for expression in stripes 2 and 3, but a discrete stripe 7 element has not been located, probably because it overlaps or shares binding sites with the stripe 2 element [I15]. The dependence of normal eve expression on wildt ~ gap gene products is almost certainly direct, as binding sitesfor the hb, Kr, and gt proteins have been found within the ~ bp stripe 2 element. As it was previously not believed that pair-rule genes respond directly to maternal polarity genes, the discovery of bed binding sites in the stripe 2 element was unexpected [112], A number of the hb and bed sites were found to overlap Kr or gt binding sites, suggesting the possibility of a role for competitive binding in eve expression within the stripe 2 domain [112], Direct evidence for this mechanism was obtained from transient cotransfection as,sam in which sequences containing these overlapping binding sites were placed upstream of a basal promoter fused to the CAT coding sequence [115], The modest synergistic activation of transcription by the combination of ~ d and hb expression plasmids could be blocked by either Kr or gt exp~ssion plasmids, These results are consistent with the fact that stripe 2 lie,,, entirely within the hb and bed domains, and with the coincidence of the borders of the stripe 2 domain with the anterior border of the Kr domain and the posterior border of the gt domain [ 112]. Competitive binding was proposed to underly the local repression of regionally activated transcription in the stripe 2 domain, and different combinations of binding sites for activators and repre,~ors within stripe elements could explain the autonomous function of the stril~ 2 and stripe 3 elements [111,112,115], in addition, the unusual properties of the hb protein ~ an activator at moderate concentrations but inactive at high as well as low concentrations ==. may play a role in the generation of stripe 3 [133], The autoregulatory element in the eve gene identified in the promoter dissection studies lies between appro0t, ~ 5,9 and ~ $,2 kb upstream of the transcription start site [39A3|, Within this sequence a 100 bp restriction fragment was identified that can mediate e,w positive autoregulation in the context of a heterolo8pus promoter construct [62], Other sequences within the ~ 5,2 to = 5,9 fragment probably have a function in autoregulation as well, because the expression directed by the lf~ bp sequence was weaker than that directed by the 03 kb sequence [62], As in flz autoregulation, ther~ seems to be a role for tissue.specific factors in e,~ autoregulation, as there is a preferential loss of ectodermal ece expression in eve- embryos [34], and the e ~ i o n of reporter genes mediated by the autoregulatory element in heterologous promoter constru¢~ is predominantly mesodermal [39,43,49,62]. Binding sites for two proteins present in embryo nu-

clear extracts, and for the eve protein itself, occur within the 100 bp element. One site is probably bound by the GAGA-factor [62], and another has been shown to be bound by the tramtrack (ttk) protein (Ref. 97; and see below). Inferences about gene interactions based solely on the results of analyses of expression of a particular gene in mutant backgrounds are problematic in that there is no way to determine whether observed effects are direct. A variety of approaches, including promoter dissection, DNA binding, transfection of cultured cells, and drug injection, have provided strong evidence for the direct nature of some of these interactions. The striped pattern of flz expression appears to develop ~lrough a general activation of flz transcription ~two~,ghout the embryo and local repression at the poles and in the interstripes ([17,28,122], and see Ref. 15 for a review). The compact zebra element is sufficient to mediate these antagonistic processes, and interstripe repression seems to depend at least in part on direct interaction between the h protein and the zebra element. The eve pattern is more directly dependent qn the functions of the gap genes, and apparently on the bed protein. However, at least with respect to the stripe 2 element, the principle of regional activation and local repression seems to apply. There is evidence for the role r,~ competitive binding interactions in the generation of this stripe, but it is not yet known what mechanisms under!y the generation of the other etw stripes.

V. An in vitro approach to identify Drosophila transcription factors The design of the massive genetic screens of Nii~Ivin-Volhard and her colleagues allowed for the identification of all zygotic lethal mutations which would produce defects in the larval cuticle attributable to disruption of processes of pattern formation [65,83,84,125]. Such screens clearly would not identify genes with a very general function, such as those encoding fundamental metabolic enzymes or basic transcription factors. Nor would those genes be detected which, when mutant, cause lethality prior to cuticle formation, or which result in internal defects not reflected in the cuticle. ~3ther classes of genes that would not pass the selection criteria would be those having a critical function at a point in the life cycle in addition to embryogenesis, or those whose wild-type function can be supplied by maternal expression (eg., caudal [78]) or redundant genes. Finally, mutations causing subtle cuticle defects might be overlooked, and ones causing gross, unclassifiable defects might mask a function in pattern formation. The ability to prepare nuclear extracts from

87

Drosophila embryos that are transcriptionally active and that contain a variety of DNA binding proteins has been seen as a way to identify trans-acting regulators of pattern forming genes that might have escaped detection by genetic screens (reviewed in Ref. 8). A particular advantage of this approach is that it is possible to prepare extracts from developmentally staged populations of embryos, thereby allowing the detection of changes in concentrations or activities of trans-acting factors during the course embryogenesis. While stagedependent differences in the transcription initiated from several promoters have been reported (see below), it has been difficult to attribute these differences to the influence of particular factors. Embryo nuclear extracts have been more useful in DNA binding assays to identify binding sites for potential trans.regulatory factors, Once a site has been identified, its sequence can be used to isolate cDNAs encoding the binding activity from phage expression libraries. Alternatively, binding assays can be used to follow a protein through purification, and expression librm3t screens can be performed using either antibodies prepared against the pure protein, or with degenerate oligonucleotides prepared through knowledge of the protein's primary sequence. All three approaches have been used successfully as described below. Transcription and binding studies using nuclear extracts from cultured Drosophila cells have been used to characterize factors interacting with a number of promoters, including those of the histone H3, H4, and the actin 5C genes [91], several of the heat-shock genes [92,127,128,129,130], the Alcohol dehydrogenase ( Adh ) gene [47], and the ecdysone inducible E74 gene [120]. Ref. 49 reported the first use of transcriptionally active embryo extracts, although extracts of whole emb~yos had been shown to be competent for chromatin assembly [37,80,81]. In addition to investigating general reaction parameters and demonstrating the fidelity of transcription initiation in the extracts, Ref. 49 provided evidence through promoter competition assays that functionally homologous transcription factors exist in mammalian and insect nuclei. Moreover, proteins interacting with the TATA boxes of several genes had been identified in cultured Drosophila cells [91,129]. Indeed, recent studies indicate that probably all of the general transcription factors first characterized in mammalian systems also exist in Drosophila. Fractions of embryo extracts can substitute for their mammalian counterparts in reconstituted in vitro transcription assays using either Drosophila or mammalian promoters [124]. Furthermore, the isolation of the genes encoding the Drosophila and human TATA-box binding proteins reveal a remarkable degree of sequence conservation, and the bacterially expressed cloned proteins can substitute for one another in reconstitution assays [54,79]. It also seems that certain promoter-specific factors may

be shared by mammalian and Drosophila cells. For example, tile Drosophila homologs of the mammalian Jun and Fos proteins have been purified from embryo nuclear extracts using the API binding sites present in the SV40 enhancer [93,94]. Not only does the Drosophila AP1 have DNA binding an~ transcriptional activation properties in common with mammalian AP1, but the two proteins are antigenically related. A detailed analysis of the en promoter using 2-12 h embryo extracts revealed the presence of eight binding sites within 400 bp upstream of the transcription start site [116]. Identified within seven of the binding sites was a sequence resembling GAGAG, and footprint competition assays using oligonucleotides containing this sequence demonstrated that the same protein bound these seven sites. The recognition sequence of this protein inspired the name 'GAGA-factor', and GAGA-factor binding sites have since been identified in the promoters of a number of genes (see below). Deletions that progressively removed GAGA-factor binding sites resulted in stepwise reductions in ,ranseription, as assayed in vitro as well as in transfected Schneider 2 cells. While no binding sites were detected downstream of the transcription start site., these sequences were also shown to be important for efficient transcription [ 116]. Nuclear extracts prepared from staged populations of embryos were used in studies of the Alcohol dehydrogenase (Adh) and Ubx promoters [5,48]. As was the case with the Adh promoter [48], the pattern of Ubx transcription was broadly reproduced in vitro in a manner dependent on upstream sequences [5]. However, changes in the binding of any single factor could not be correlated with the pattern of transcription. Three of the five footprints within 200 bp upstream of the start site were attributable to binding by the GAGA-factor. A purified 67 kDa protein was shown to possess the GAGA-factor binding activity, and addition of the pure protein to a cytoplasmic extract that supports basal levels of transcription and which has minimal endogenous GAGA-factor binding activity stimulated transcription of wild.type, but not truncated Ubx templates [5]. The product of the zeste gene also binds multiple sites in the Ubx promoter, some of which overlap GAGA-factor sites, and purified zeste protein was able to activate Ubx transcription in cytoplasmic extracts as well [6]. Several footprints were also detected downstream of the transcription start site, and a dependence on downstream sequences for efficient transcription in nuclear extracts was shown. Several of these sites are A-T rich, resembling borneo-domain binding sites upstream of the en transcription unit, and are known to bind a number of borneo-domain proteins, In a subsequent report, it was shown that binding of the eve protein to one of these footprinted downstream sites mediated repression of Ubx

88 transcription, whereas the zen protein bound to the ~me site had no effect [7]. in vitro transcription assays provide the most direct method to establish the function of a putative tran~ription factor, and the demonstration that eve protein can function as transcriptional represser in such a system is consistent with the conclusion drawn from eotransfeetion experiments (see above), The ol protein has also been shown to function as a represser in vitro, aRhough the mechanism was unexpected: the protein was found to bind the TATA box (which resembles a consensus home-domain binding site) and prevent transcription by bk~k, the binding of TFIID [r~]. AI" though the physiological significance of this finding is uncle~,r, it suggests an intriguing mechanism of gene control. The U ~ protein has also been shown to function as an activator in vitro [64]. A DNAoaffinity fraction of embryo nuclear extract that is responsible for binding to one of the upstreana sites in the Ulna' promoter was independently shown to bind a site within a small region, termed element !, previously identified as being required for neuronal oxpregsion of the l~padecarboxylase (D de) gene [9]. Ddc is expressed in the embryonic and larval central nervous gyglems, and at late stages of embryogenesis, it is expressed in the epidermis where it i~,,ctions in hardening of the cuticle. Element ! was implicated in neuronal, but not epidermal, Ddc expression [9,10,107]. A factor that binds element I, called EIf-I [10], was shown to correspond to the same factor (called NTF-I) that binds the Ubx promoter [27], and evidence that EIf-I/NTF-I can function as a sequence-specific activator of transcription was obtained by adding the purified protein to depleted nuclear extracts [27], Using a monoclonai antitardy [10], or primary sequen~ information [27!, cDNAs encoding the Eifi / ~ F , . I binding activity were obtained, Characterizalion of th¢,~ cDNAs indicated that EIf-I/NTF-I consists of a family of proteins arising from alternative RNA splicing, and that the proteins share a potential glutamine-rieh activation domain along with a sequence related to the helix-loop-helix DNA binding domains of MyoD and myogenin, Analysis of the emb~onic expression of EIf-I/NTF-! transcripts by in situ hybridization revealed that they are expressed throughout the ectoderm by 5~5,5 h and, subsequently, in clusters of ~lls in the brain and ventral nerve cord [271, Genetic analysis"'of the gene encoding EIf-I/NTF-I [il] and a detailed di,~ction of the Ddc promoter region [1~] preclude simple models for the function of EIf,.I/NTF-I in the neuronal expression of Ubx and ~&', Embryonic lethal mutations in the EIf-I/NTF-I gene were shown to correspond to mutations in the previously identified gene grab~vhead (g~l). Embryos mutant for grh have a fragile cuticle and exhibit de-

fects in the head skeleton, although segmentation appears normal. Surprisingly, neither neuronal Ddc expression, nor element I-mediated expression of a reporter construct, were detectably abnormal in grh mutants. Neuronal expression of Ubx was similarly normal in these mutants. These results clearly indicate that EIf-I/NTF-I is not essential for the neuronal expression of Ddc, despite the fact that the site to which it binds is essential. A possible explanation for this apparent contradiction is that a factor distinct from Elf- I.NTF- I, but which binds the same site, can activate neuronal Ddc expression [11]. Furthermore, results of a recent, more detailed analysis of the Ddc promoter support the existence of an additional positive regulatory element immediately upstream of Element i, and also suggest the possibility that, in additiun to Elfol/NTF-I, a negative transcriptional regulator may bind Element ! [1081. Another perplexing result reported by Ret\ 11 is that while neuronal Ddc expression was unaffected in grh mutants, epidermal expression was abolished. Element | activity had previously been shown to be highly specific for neuronal expression [9,11,63,107], and disruption of the Elf-I/NTF-I binding site in the context of the entire Ddc regulatory region had no effect on epidermal expression [9]. Previously there was only circumstantial evidence for a role for EIf-I/NTF-I in regulation of epidermal Ddc transcription, in that the two genes are co-expressed in this tissue during late stages of embryogenesis [27]. it is possible that other binding sites for EIf-l/NTF-I exist in the Ddc gene, and that the element I site is either irrelevant or redundant. Perhaps the most salient point made by the results of Ref. I I is that although an in vitro approach may allow the identification of potential trans-activat. ing factors, their in vivo function may not be a simple as biochemical results suggest. As discussed above, the ftz regulatory region has been dissected using P-element transformation assays, leading to the identification of three functionally dis. tinct elements. The promoter proximal zebra element and the upstream enhancer-like element have been the subjects of biochemical analyses using embryo extracts and purified horace-domain proteins [12,17,18,44,45, 95,121-1231. DNAase I protection assays of embryo extracts revealed a large number of binding sites within the 2.6 kb upstream element [44]. One of these which showed stage-specific patterns of protection was then found to be bound by a protein encoded by a previously unidentified gene which was given the name tramtrack (ttk, [45], and see below). Also identified were several sites that are A-T rich and resemble those bound by homeo-domain proteins. As part of the functional dissection of the upstream element described above, Ref. 95 identified a large number of binding sites for the ftz homeo-domain, a finding consistent

89 with the autoregulatory function mediated by the element. The Eif-I/NTF-1 protein also binds multiple sites in the upstream element [27]. Multiple binding sites for two proteins have also been identified within the ftz zebra element, two of which arc bound by the ttk protein [12], and two by a protein named FTZ-FI [123]. These sites are within elements having both positive and negative regulatory properties as defined by Refs. 17 and 18. Consistent with the idea that these elements contain closely apposed or overlapping binding sites for trans-acting factors with opposite effects on ftz transcription, P-elem e n t transformation assays of zebra element function showed that mutation of the ttk binding sites resulted in both premature and ectopie transcription [12], while mutation of the FTZ-FI sites resulted in reduced levels of zebra element-directed transcription [123]. As one of the ttk protein binding sites overlaps a FTZ-FI site, it is possible that competitive binding between these two proteins is involved in some aspect of ftz regulation. The structure, expression, and function of the ~tk protein are discussed in more detail below. A eDNA encoding the FTZ-FI protein has been cloned, and its sequence reveals that the protein bears homology to mammalian steroid receptors, having a ligand binding domain and a pair of zinc fingers of the C.,C., class

[711. Embryo nuclear extracts have also been used in an analysis of the ece promoter [92]. It was found that efficient transcription in vitro depends on sequences upstream of the transcription start site that contain a number of overlapping GAGA-factor binding sites. In addition, a binding activity that is developmentally regulated recognizes four sites within 500 bp upstream of tl~e start site as well as a single site within the 100 bp ece autoregulatory element [96,97]. The sequence of a eDNA encoding this binding activity indicates that it corresponds to the ttk protein [97]. The identification of multiple ttk protein binding sites within the autoregulatory elements and promoter-proximal regions of both eve and ftz raises the possibility that the ttk protein performs a similar role in the regulation of these two genes (see below). Finally, while the responsible protein was not characterized, an additional binding site was identified approx. 50 bp downstream of the transcription start site. in addition to the en, Ubx, and ere promoters, GAGA-factor binding sites have been identified within upstream sequences of a number of other genes, ineluding Antp (discussed in Ref. 93), the ecdysone-inducible E74 gene [120], several heat-shock genes [38], and within the ftz zebra element [122]. Binding sites also occur within the intergenic region of the divergently transcribed histone H3 and H4 genes [38], as well as both upstream and downstream of the Kr transcription start site [67]. In the context of super-

coiled plasmids, sequences within the hsp26, hsp70, and the H 3 / H 4 promoters have been shown to be sensitive to SI nuclease digestion at low pH, indicating deviation from the B-form DNA conformation [38]. DNAase ! lootprinting assays in intact nuclei revealed that these SI sensitive sites were bound by a protein that was subsequently shown to most likely correspond to the GAGA-faetor. A model was proposed in which it was suggested that these promoters are maintained in a state ir~ which they are 'poised' for transcription, and that the GAGA-factor has a role in maintaining this poised state [38]. Consistent with this idea, it has since been shown that the GAGA-factor does not possess true activation function, but rather that it is capable of counteracting repression by historic H! [16,67]. VI. The ta}le of tramtrack in eve and ftz regulation As an example of a gene identified exclusively by biochemical methods, and which is likely to play an important role in embryogcncsis, wc will discuss in some detail the ttk gene. As mentioned above, the ttk protein was initially discovered in a biochemical analysis of the flz promoter. Binding sites for a number of proteins present in embryo nuclear extracts were identified within the ftz upstream enhancer element [44]. A 2.8 kb eDNA encoding a protein that bound one of these sites was obtained by screening an expression library with an oligonueleotide containing the bound sequence [45]. The protein encoded by this eDNA has a theoretical molecular weight of 69 kDa and contains two zinc finger motifs of the C , H 2 class. Analysis of the embryonic expression of the corresponding mRNA has been performed by in situ hybridization [45,97]. The mRNA is present throughout the embryo during the syncitiai cleavages, but disappears by the cellular blastoderm stage. Following gastrulation, the mRNA is detectable in several tissues, notably the anterior and and posterior midguts, and very late in embryogenesis, it is present throughout the epidermis. The concurfence of the initial disappearance of presumed maternal ttk transcripts with the onset of ftz transcription led to the proposal that ttk may function as a repressor of ftz transcription [45]. Independently, a eDNA encoding the ttk protein was isolated by virtue of the protein's ability to bind two sites within the fiz zebra element [12]. A functional analysis of these sites was perlormed by P-clement transformation with zebra element-lacZ artificial promoter constructs. It was found ihat mutations of the ttk protein binding sites within the zebra element resulted in the detection of ~-galactosidase as early as the third nuclear cleavage. In lines transformed with the wild-type zebra elementdacZ control construct, /J-galactosidase was not detected before the ninth nu-

pre - eb

cb

gbe

dye

~

nk

Fii~. 2. Major features of the expression of el.e. ft:. and ~'tk in early stages of ¢mbryogenesis. Expression of the protein products of the genes are depicted during three stag.¢s of ¢mhryogcnesis to show possible regulatory relationship between tek and ere and ftz in early stages and apparently unrelated late patterns of expression, Emh~os are oriented anterior to the left and dotal to the top, Maternally supplied irk protein is detected throughout mo~t of the pre..cellular blastoderm stage (pre=cb). prior to the onset of e~.e and fi: expression. The disappearance of the maternal , k protein just prior Io cellularization coincides with the initiation of e~'e and f¢: protein expression at low levels in broad regions of 1h¢ embryo. No ztk protein is detcch~bl¢ in the cellularizcd embryo (cb). by wllich time the e~'e and fiz proteins are expressed in complementary patterns el' ~vea sharply re~lved transver.~ stripes. By the la,er stages of germband elongation (gb¢). the seven flz stripes and the seven major and sevenminore.~t~stripes di:sapl~arand both proteins are now expres~d in the extreme posterior end of the germband: zygotic expression of ttk is d~t¢cted in the anterior midgut invagination and within the scattered nuclei of the yolk sac, In subsequent stages, e~¢ and fiz expression is restricted to nuclei of the nervous system and uk comes to be expressed throughout the gut and epidermis.

clear cleavage. At the cellular blastoderm stage, the mutant, but not the wild-type z e b ,-element directed lacZ expression at high levels throuhaout tffc embryo. Thee results demonstrate the functional significance of the two ttk binding sites in the ftz zebra element, and suggest a role, if not for the 69 kDa ¢¢k protein, then for a protein with a similar binding specificity, in the regulation of ftz transcription. The results of Ref, 12 also show that the Drosophila e m b ~ is transcriptionally competent very ,s~n after fertilization, This finding was somewhat surprising, as v¢~ little transcription of any class of gene is detected prior to the ninth nuclear cleavage [3,29,69], and it has been propo~d that an essential component of the basic transcriptional apparatus is n0~ synthesized until that time [~], Ref, 12 proposed that a fixed amount of maternally supplied 69 kDa ttk protein functions to pre~nt the premature transcription of ftz, The exponential increase in the number of ftz promoters resulting from the synchronous precellular nuclear cleavages ~ l d have the consequence that by the ninth cleavage the amount of ttk protein per promoter would become limiting. Relief of repre~ion by the ttk protein would then allow activation of f¢~. transcription by gap and pair-~le genes, While this model is attractive for its simplkity, it should be pointed out that other mechanisms for eliminating the uk activity at the appropriate time are also possible, These might include control at the level of translation, message stability, or degradation of the 69 kDa protein, In ,dew of the severe

disruptive effects resulting from ectopic expression of segmentation genes [58,59,118], the added level of regulation afforded by such a mechanism might be critical, especially in the case of genes such as ft. and eve, which have positive autoregulatory activity [12]. Tile identification of a protein present in embryo extracts that binds sites within the promoter region and autoregulatory element of the eve gent as the 69 kDa ttl: protein suggests a possible role for ttk in the regulation of this gone as well [96,97]. Fig. 2 compares the pattern of expression of ttk with the eve and ftz patterns during critical stages of embryogenesis to indicate that possible regulatory relationships exist only at early times, consistent with the notion that maternal 69 kDa ttk is a repressor of these two genes. Additional support for the idea that maternally supplied 69 kDa ttk protein represses ere and ftz transcription in the cleavage stage embryo was obtained through the use of transgenic embryos expressing the ttk eDNA under the control of the hsp70 promoter [97a]. It was shown that ectopic expression of the 69 kDa ttk protein resulted in the loss of both eve and ftz stripes, especially in the middle of the embryo. Ectopic expression was lethal to the embryos, and at early times resulted in severely deformed cuticles, including ones which lacked evidence of segmentation. Ectopic expression at later times gave rise to embryos that had apparently failed to undergo germband retraction. The severity of the cuticle defects observed, along with the variety of processes that seemed to be affected, suggest that ttk is

91 involved in controlling the expression of other genes in addition to eve and flz. These findings, coupled with the identification of a second form of the ttk protein arising from alternative splicing and containing DNA binding Zn 2+ fingers with a different binding specificity [97], suggest a complex role for ttk, and offer an explanation for its absence from genetic screens. VII. Summary and Perspectives Most of the genes involved in pattern formation in

Drosophila have been identified through genetic screens, and the regulatory interactions between them have been elucidated through a combination of molecular and genetic techniques, The discovery that most of these genes function within this regulatory network by controlling the transcription of other genes, along with the ability to locate cis.regulatory dements within a gone by P.olement transformation of reporter gone constructs, have contributed significantly to understanding eukaryotic transcriptional regulation. The identification of so many cis- and trans-regulatory elements in such complex regulatory systems would not have been possible in an organism that did not have the history of genetic analysis that Drosophila has. Complementing these studies in understanding the mechanisms of genetically identified regulators have been experiments that have employed cultured Drosophila cells and transcriptionally active embryo nuclear extracts. Such extracts have also led to the identification of additional regulatory proteins not previously identified by genetic methods. It is likely that this powerful combination of genetic, molecular and biochemical approaches will continue to provide fascinating insights into the mechanisms by which complex regulatory cascades contribute to the control of ¢mbryogenesis. References I 2 3 4 5 6 7 8 9 10 11 12 13

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