Coming to grips with cactus

SHUBHA GOVIND AND RUTH STEWARD

GENE REGULATION

Coming to grips with cactus Dorsal is a transcription factor that regulates Drosophila embryonic polarity. It is cytoplasmic when associated with cactus protein ‘until extracellular signals cause its release and nuclear entry. Embryonic axial polarity in Drosophila is controlled by basic cellular mechanisms: signal transduction, subcellular localization of detemAants and transcriptional regulation. The protein ‘dorsal’ is the morphogen responsible for establishment of the dorsoventral axis. The gene c&w Sal is so named because mutations in it result in an embryo that has lost its dorsoventral asymmetry so that ventral regions resemble normal dorsal regions (a ‘dorsalized’ phenotype) - suggesting that dorsal normally functions in the ventral and lateral regions of the embryo.

Dorsal is a member of the rel family of transcriptional regulators, and can function as either a transcriptional activator or a repressor. The ability of nuclear dorsal protein differentially to control the expression of target zygotic genes as a function of concentration means that a gradient of dorsal protein can confer positional identities along the embryonic dorsoventral axis. Dorsal protein is laid down in the egg before fertilization; the protein is distributed uniformly throughout the

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Cactus

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Extracellular

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Fig. 1. (a) The establishment

of embryonic dorsoventral polarity. The ted to the embryo by a signal transduction pathway. Binding of an signal is transduced through the cytoplasm by the tube gene product and/or cactus, leading to dissociation of the dorsal-cactus complex on its interaction with dorsal. (b) In an analogous way, arrival of a NF&-lxB complex, possibly through the actions of a protein kinase it regulates transcription. It is likely that 1x9 is also stabilized by the

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asymmetry defined in the egg chamber during oogenesis is transmitextracellular ligand activates the Toll transmembrane receptor. The and by the pelle protein kinase which may phosphorylate dorsal and nuclear translocation of dorsal. Stability of cactus is dependent cytokine at the surface of a lymphocyte causes dissociation of the that phosphorylates MB, fredng NF-x9 to enter the nucleus where NF-x9 heterodimer.

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cytoplasm of early embryos and this distribution has been postulated to arise as a result of a specilic and direct interaction between dorsal and a second protein, cactus [ 1,2]. Dorsal moves from the ‘cytoplasm to the nuclei at the syncytial blastoderm stage of embryonic development, again in a ventral-to-dorsal gradient. The gradient of nuclear dorsal protein arises in response to an extracellular signal that is synthesized, controlled, and mediated by at. least ten other gene products of the ‘dorsal group’ (Fig. 1). Mutation of any of these genes causes a dorsalized phenotype. According to the current model, seven genes of the dorsal group act ‘upstream’ of Toll, a transmembrane receptor, to generate an extracellular ligand that binds to and activates the Toll receptor on the ventral and lateral sides of the embryo. Graded activation of Tsoll from the ventral to the dorsal side of the’embryo would result in transduction of this signal, through the cytoplasm, by way of the functions encoded by the genes tube and pelle. Phosphorylation of dorsal, or of both dorsal and cactus, results in the asymmetric nuclear targeting of the dorsal protein after dissociation of the cytoplasmic dorsal-cactus complex. Free dorsal protein is then released to ventral and lateral nuclei; free cactus protein is unstable and may be degraded. The gradient of nuclear dorsal protein controls transcription and. initiates the program of zygotic gene expression that ultimately determines cell fates along the dorsoventral axis. The intracellular events of signal transduction that lead to the nuclear import of previously cytoplasmic dorsal protein are similar to those that regulate members of the vertebrate rel family of transcription factors - including p50 (NF-%Bl) and ~65 (RelA), the subunits of the transcription factor NF-xB; RelB; c-r-e1and the oncogenic v-rel [3]. The rel proteins share a 300 amino-acid ‘rel homology domain. Their nuclear import is inhibited in a variety of cell types by members of a second family of proteins, IxB -which includes I%Btx (pp4O/MAD-3), I%Bj$ and the human proto-oncogene product bcl3. An extracellular signal generated by a cytokine, mitogen or virus releases the IzBdependent cytoplasmic retention of the rel subunit of the rel-I%B complex, allowing rel to be translocated to the nucleus. Interestingly, the interleukin-1 receptor and Toll have significant sequence similarity in their intracellular domains. The recent molecular cloning and characterization of the cactus gene has made clearer the similarity between the two pathways, as cactus is now identified as a member of the I%B family, Two groups have used different approaches to cloning cactus: Geisler et al. [4] used a functional approach whereas Kidd 151 used ‘reverse genetics’. The identity of the cactus clone was confirmed by rescue of the cactus mutant phenotype by injection into the embryo of RNA from the cloned gene, the use of antisense RNA to induce phenocopies of the cactus mutation, and by molecular mapping of an insertion mutation in the cactus gene.

The cactus locus is transcribed from the maternal genre as a 2.1 kb RNA strand and from the zygotic genome as a 2.3 kb RNA strand; the zygotic protein is predicted to be I8 amino acids shorter than the maternal. The sequences of the cactus and IxB proteins are similar in organization. The cactus proteins are predicted to encode an acidic, cytoplasmic protein with six ankyr& repeats (Fig. 2). The amino-terminal domain of each is highly acidic, whereas the carboxy-terminal domains contain PEST (Pro-Glu-Ser-Thr) sequences characteristic of unstable proteins. Ankyrin repeats have been found in various regulatory proteins such as cdcl0 and SWIG, which are involved in cell cycle regulation, and lin-12, glp-1, and Notch, which function in cell fate detertination during development. The repeats are thought to function in protein-protein recognition and possibly, as in the transcription factor GABPj3, in DNA binding j.61. Maternal

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Fig. 2. Structures

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maternal

and zygotic

cactus

proteins.

Two groups have studied the cactus protein ( [ 51, and A.M. Whalen and R Steward, unpublished data). Consistent with finding two ~RNA~, maternal and zygotic cactus proteins have been identified by Western blotting, with molecular weights of approximately 72 kD and 69kD, respectively. The maternal cactus protein is associated with dorsal protein in extracts of ovarian and embryonic cells; it is exclusively cytoplasmic, suggesting that dorsal dissociates from cactus before migrating to the nucleus. Cactus is also found in the cytoplasm of the follicle cells that surround the egg in the egg chamber. The linding that cactus is a cytoplasmic protein is somewhat at odds with the observation that the interaction of cactus with dorsal inhibits the binding of dorsal to its target DNA sequence [4,5], which would seem to imply that cactus might also enter the nucleus and function there to regulate the DNA-binding activity of dorsal. Several lines of evidence suggest that a homeostatic mechanism, which regulates the absolute levels of dorsal and cactus proteins, controls the proper patterning of the embryo ([7], and AM. Whalen and R. Steward, unpublished data). Both dorsal and cactus genes show

DISPATCH

haplo-insufficiency where one dose of either dorsal or cactus can result in a partial loss-of-function phenotype, suggesting that the absolute level of neither protein is in excess. The finding that cactus inhibits the selective nuclear import of dorsal by al direct interaction suggests that over-expression of dorsal within the embryo should saturate the available maternal cactus protein. After all maternal cactus protein is saturated by dorsal, extra dorsal will be free for nuclear import resulting in a ventralized phenotype. This phenotype was indeed observed with greater than three copies of dorsal. This means that the coding potential of cactus is high enough to inhibit only some extra dorsal; once all the maternal cactus transcript is translated and stabilized by dorsal, the embryo becomes ventralized. Furthermore, the absolute amount of dorsal protein determines how much cactus will be stabilized and therefore how much is present in the cytoplasm as dorsal-cactus complex (Table 1). Although increasing the amount of dorsal relative to maternal cactus protein to sufficiently high levels can cause ventralization, increasing the cactus gene to three times the amount of dorsalgene has no effect on patterning in the embryo. Because free cactus protein is unstable, it is unlikely to be a target of the dorsal group signal that results in nuclear import of dorsal. Instead, this signal must modify either components of the dorsal-cactus complex, or free dorsal alone. In the dorsal-cactus complex in the oocyte or embryo cy toplasm, both proteins are phosphorylated ([5], and A.M. Whalen and R. Steward, unpublished data). No change to the phosphorylation state of the maternally contributed cactus is evident during (early embryogenesis, when the complex dissociates, nor is cactus phosphorylation altered by mutations in dorsal group genes, which cause the dorsal-cactus complex to dissociate. Thus, phosphorylation of cactus appears not to be involved in dissociation of the complex -- but the possibility cannot be ruled out, because the stabilization of cactus by dorsal

Maternal of ‘cactus

means that uncomplexed cactus might be unstable and its phosphorylation state could therefore not be assessed. By contrast, the phosphorylation state of dorsal protein does alter during embryogenesis. Dorsal is found as a single phosphorylated form in extracts of ovarian tissue. In the early embryo, several phospholylated forms are found and they are differentially &ected by mutations in the dorsal group of genes and cactus (A.M. Whalen and R. Steward, unpublished data). One of these phosphory lated isoforms is absent from dorsalized embryos but is more abundant in ventralized embryos than in wild type. Analysis of the sequence of pelle, which has recently been cloned by Shelton and Wasserman [S] , indicates that it encodes a protein serine/threonine kinase of the raf/mos family; the catalytic activity of the kinase was shown to be required for rescue of the loss-of-function pelle phenotype. It is therefore possible that dorsal is a direct substrate for the pelle protein kinase. Activation of pelle in response to an extracellular signal at the Toll receptor would then cause phosphorylation of dorsal, or possibly cactus (Fig. 1) and its dissociation from cactus. Unlike NF-xB, which consists of a heterodimer of p50 and p65 subunits, dorsal functions as a homodimer [9]. Cactus also seems likely to function independently of any other protein of the rel family as dorsal alone stabilizes cactus. It remains possible that zygotically encoded cactus is stabilized by another rel protein that is expressed later in development. In vitro deletion analysis of cactus has shown that the dorsal-binding region coincides with the ankyrin repeats [ 51. Other mutations suggest that the amino-terminal region of cactus may be involved in regulating dissociation of the dorsal-cactus complex (Fig. 2;

[31X Regulation of gene expression at the level of selective nuclear import of proteins from the cytoplasm is a recurrent theme among eukaryotes. The glucocorticoid receptor-Hsp90 (heat shock protein) complex, the yeast

copies gene

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s detectable in the ab! jet7 ce of dorsal protein (I efi t). With increasing am ounts of dorsal present; i 3re ctic&~$ is henotjipe remains will d-t ype with as many as : :opies of the- dorsal ge present. With:>3 cop s of ,dprsal (right), ,’ urated and excess dor sal produces ? ,\(entralk ed phenotype. ‘, 0 1993,‘ciJynt -

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transcription factor SWIS, and NF-AT, the transcriptional regulator of lymphlokine and cytokine genes, are some examples of this type of post-translational control of nuclear function. Sequestration of regulatory proteins in the cytoplasm and ltheir subsequent nuclear import, triggered by the arrival of extracellular signals, allows cells to achieve rapid and specific activation of transcriptional regulators in response to signals from the outside.

4.

tus,

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6.

7.

References 8. 1.

2.

3.

ROTH S, Hmoim

Y, GODT

D, NUSSLEIN-VOLHARD

C: cuctus,

a ma-

ternal gene required for proper formation of the dorsoventral morphogen gradient in Drosophila embryos. Development 1991, 112:371-388. GOVIND S, STEWARD R: Dorsoventral pattern formation iu Drosophila signal transduction and nuclear targeting. Trends Genet 1991, 73119-125. BUNK V, KOURILSKY P, ISRAEL A: NP-xB and related proteins: rel/dorsal h~omoIogies meet ankyrin-like repeats. Trends B&hem 1992, 17:135-140.

GEISLER R, BERGMANN

9.

A, HIROMI

Y,

NuSSLEIN-VOLHARD

C: wG

a gene

involved in dorsoventral pattern formation of Drosophila, is related to the IxB family of vertebrates. Call 1992, 71:613-621. KIDD S: Characterization of the Drosophila cactus locus and analysis of interactions between cactus and dorsal proteins. Cell 1992, 71: 623435. MICHAELY P, BENNFTI V: The ANK repeat: a ubiquitous motif invoIved in macromolecular recognition. Trends Cell Biol1992, 2:127-129. GOWVD S, BREXNAN L, STEWARD R Homeostatic balance between dorsal and cactus proteins in the Drosophila embryo. Development 1993, 117~135-148. SHELTON CA, WA.SSERMAN Sk pelle encodes a protein kinase required to establish dorsoventral polarity in the Drosophila embryo. Cell 1993, 72:515-525. GO~IND S, WHALEN AM, STEwARD R In vivo self-association oi the dorsaI protein of Drosophila. Proc Nat1 Acad Sci C’SA 1992, 897861-7865.

Shubha Govind and Ruth Steward, Department of Molecular Biology? Princeton University, Princeton, New Jersey 08544, USA

THE AUGUST 1993 ISSUE OF CURRENT OPINION IN GENETICS AND DEVELOPMENT will contain the following reviews on Pattern formation and developmental mechanisms, edited by Peter Gruss and Bill McGinnis:

Homeotic genes by G Morata Neural fate specification by J Modolell Segmentation in DrosopbiZu by T Kornberg Dorsal-ventral patterning in DrosopbZZu by R Steward Developmental control of splice-site selection by D Rio Neural induction in mammals by 1 Jessell and Ariel Ruiz I Altaba Early gastrulation in mammalian development by R Beddington C. eleguns homeobox gene clusters by T Burglin and Gary Ruvkun Zebrafish developmental genetics by M Mullins and C NussleinVolhard ‘Community effects’ and mesoderm induction in Xenopus by J Gurdon Control of limb patterning in the chick and mouse by C Tabin and Bruce A Morgan Transcriptional regulation and spatial patterning in DrosopbiZu by H Jackie and Michael Hoch Germ-cell specification in Drosopbdu by P MacDonald and Joan E Wilson Neural specification/neural crest in vertebrates by M Bronner-Fraser Cell-cell interactions in cell fate decisions by S Artavanis-Tsakonas Spemann organizer factors in Xenopus by J Smith Paired domain genes in DrosopbZZa by M Noll Mouse Hox genetic functions by R Krumlauf