BIOLOGICAL OVERVIEW

brinker is a potential transcription factor that negatively regulates decapentaplegic target genes (Bray 1999). brinker was independently characterized as a modifier of dpp signaling in three laboratories (Campbell, 1999; Jazwinska, 1999a, and Minami, 1999). In an enhancer trap screen designed to identify genes whose expression may be regulated by Dpp in the imaginal discs (Campbell, 1999), one potential line, X47, was selected that appeared to exhibit marker gene expression at the anterior and posterior extremes of the wing disc but not in the central region. Dpp is expressed along the compartment border within this central region, suggesting that the gene marked in the X47 line may be negatively regulated by Dpp signaling. In Jazwinska (1999a), a collection of 3200 X-linked lethal mutations was screened. One mutant was identified that showed a strong expansion of the dorsal epidermis and concomitant reduction of the ventral epidermis, indicating a defect in Dpp signaling. This indicated that brk negatively affects either the distribution of Dpp, the reception of the Dpp signal, or the activation of Dpp target genes. Minami (1999) isolated brinker by characterizing two strains with P-element insertions that generate expression patterns complementary to those of Daughters against dpp in the wing disc. The two strains have P-element insertions in polytene cytological location 7B, a region to which brinker had been genetically mapped by Roth and Wieschaus laboratories in the first two studies.

An abundance of attention has been paid to proteins which, together with the SMADs, act as activators of transcription downstream of Dpp. However, one case is known from C. elegans where activated SMADs counteract the specific repression exerted by a DNA-binding inhibitory SMAD (Patterson, 1997 and Thatcher, 1999). In addition, analysis of Drosophila Brinker suggests that one purpose of SMAD signaling is to antagonize transcriptional repression of Dpp target genes (Campbell, 1999; Jazwinska, 1999a and b, and Minami, 1999). In the wing, the Brinker protein acts as a repressor of low and intermediate level Dpp target genes. These genes are expressed upon loss of brk even in the absence of SMAD signaling, suggesting that Brk acts as a direct transcriptional repressor. Since brk expression itself is negatively regulated by Dpp, target gene activation, at least in part, results from downregulation of brk.

This overview will examine the role of Brinker in regulating Dpp target genes in the early zygote. Consistent with brk’s role in the wing disc, embryonic target gene activation in the absence of brk is independent of SMAD activity. Thus, in both contexts, brk acts either parallel to or downstream of SMADs as a specific repressor of low and intermediate level Dpp target genes. brk is expressed like another dpp antagonist, short gastrulation (sog), in ventrolateral regions of the embryo abutting the dorsal dpp domain, and in brk mutants dpp expression expands to cover the entire ectoderm. In this situation sog is largely responsible for Dpp gradient formation, since brk;sog double mutant embryos have almost no polarity information in the ectoderm. The double mutants consist mainly of mesoderm and unstructured dorsal epidermis. Thus, brk and sog together specify the neuroectoderm of Drosophila embryos (Jazwinska, 1999b).

The Dpp activity gradient that is established in the dorsal half of cellular blastoderm embryos subdivides the ectoderm into three major territories that can be identified by the cell types they will comprise and/or by the cuticular structures they secrete. High levels of Dpp activity specify the dorsalmost region, which differentiates into large amnioserosa cells that express Krüppel (Kr) protein. These cells form extraembryonic tissue that does not produce cuticle. Lower Dpp levels in the dorsolateral region specify the non-neurogenic ectoderm, harboring peripheral nervous system precursors that can be identified as small segmentally repeated snail (sna)-expressing cells. This region, the dorsal epidermis, gives rise to cuticle with weakly pigmented dorsal hairs. Finally, the ventral (neurogenic) ectoderm requires very low levels or the absence of Dpp to form the neuroblasts of the central nervous system, which also expresses sna. This region, the ventral epidermis, secretes cuticle with heavily pigmented ventral denticles. The cuticle of brk mutant embryos has an enlarged region carrying dorsal hairs and a smaller region carrying ventral denticles. The number of sna-expressing neuroblasts in the ventral neurogenic region is reduced. This indicates that brk mutations lead to an expansion of dorsolateral fates and a reduction of ventrolateral fates. However, despite these lateral fate shifts, the number of Kr-expressing amnioserosa cells is not different from wild type. Thus, brk specifically affects cell fates depending on low or intermediate levels of Dpp signaling, while those that require peak levels are not altered (Jazwinska, 1999b).

To identify the underlying causes of the visible changes in cell fate, the effect of brk was examined on the expression of two groups of dorsal/ventral (D/V) patterning genes. The first group consists of dpp, zerknullt (zen) and tolloid (tld), whose expression is initiated very early in syncytial blastoderm stages. Since they are ventrally repressed by Dorsal (Dl) protein, their expression domains are confined to the dorsal 40% of the egg's circumference. In brk mutant embryos dpp, zen and tld expression is initiated normally. However, in contrast to wild type their expression domains expand ventrally during mid-cellularization. These data demonstrate that brk is not required for the early ventrolateral repression of these genes, but is essential to prevent their lateral expansion during cellularization. Since at these stages Dl protein is still present in the lateral nuclei, brk might function to augment the Dl-mediated repression of dpp, zen and tld (Jazwinska, 1999b).

The second group of DV patterning genes includes rhomboid (rho), u-shaped (ush) and pannier (pnr), which are not direct targets of repression by maternal Dl. The initiation of their expression during cellularization requires prior formation of the Dpp activity gradient. Therefore, they are candidates for being direct targets of Dpp signaling in the embryo. They are expressed in domains straddling the dorsal midline that are 12 (rho), 14 (ush) and 32 (pnr) cells wide at cellular blastoderm (cell counts at approx. 50% egg length). The two narrowly expressed genes rho and ush are not changed in brk mutant embryos. This is also true for late zen expression, which in brk mutant embryos, as in wild type, refines to a narrow 5- to 6- cell-wide stripe along the dorsal midline despite the prior expansion. However, pnr expression expands in brk mutant embryos and low ectopic pnr levels can be seen in a broad lateral domain that stops about five cells short of mesodermal sna expression. Thus, brk does not affect the Dpp target genes that are expressed in dorsalmost regions and supposedly depend on highest Dpp levels. However, a target gene that is expressed in a wider domain, and is therefore presumably activated by intermediate levels of Dpp, is expanded (Jazwinska, 1999b).

The described alterations in DV patterning appear to result from complete loss of brk function, and deficiencies uncovering brk produce phenotypes identical to the original point mutation brk M68. This mutation contains a stop codon causing an early truncation of the protein (Jazwinska, 1999a). The phenotypes are also not enhanced in embryos derived from females in which germline clones homozygous for brk M68 have been induced, indicating that the zygotic phenotype is not ameliorated by maternal expression. In summary, brk mutations affect the Dpp activity gradient in the embryo by expanding the domains of expression of dpp and one of its activators (tld) into ventrolateral regions. Despite the uniform expression of dpp in the entire ectoderm, Dpp activity levels appear to be only mildly increased in the ventrolateral region since only low-level (zen) or intermediate-level (pnr) target genes are ectopically expressed, causing a reduction in the size of the nervous system and ventral epidermis accompanied by an expansion of dorsal epidermis. Peak levels of Dpp in dorsalmost positions appear to be normal, judging from both target gene expression and cell type differentiation (Jazwinska, 1999b).

The question arises as to why uniform expression of dpp in the ectoderm is compatible with the substantial degree of DV polarity exhibited by brk mutant embryos. In wild type, Dpp activity is polarized by sog expression in the ventrolateral region of the embryo, such that ventrolateral Dpp activities are reduced and a peak of activity is established centered on the dorsal midline. Embryos mutant for sog show a reduction of ventrolateral fates, albeit to a weaker degree than brk embryos. In contrast to brk embryos, they differentiate only a small number of scattered amnioserosa cells. The lateral fate shift is not accompanied by strong expansion of dpp transcription as seen in brk embryos, and pnr expression is not as greatly expanded as in brk embryos. In brk embryos, sog is still expressed in the ventrolateral domain of the ectoderm. To test whether this expression accounts for the DV polarity in the ectoderm of brk embryos, brk embryos were constructed that were also mutant for sog. In contrast to either single mutant, the ectoderm of brk sog embryos forms only dorsal-type cuticle hairs and completely lacks ventral denticles. During germ band extension, neuroblast expression of sna cannot be detected; instead, sna expression in the peripheral nervous system (PNS) precursors, normally restricted to dorsolateral regions, expands into the ventral region of the embryo. This suggests a complete deletion of the ventral neurogenic region (Jazwinska, 1999b).

Taken together, brk and sog have both overlapping and distinct roles in shaping the Dpp activity gradient of the Drosophila embryo. While sog has an important function in providing peak levels of the gradient necessary for amnioserosa development, brk and sog together are essential to limit the ventral extension of the anti-neurogenic activity of dpp. Interestingly, brk;sog double mutants do not completely eliminate all polarity of the ectoderm. This can be seen from the expression of pnr, which in the double mutant has uniformly high ectodermal levels except in a 5-cell wide stripe bordering the mesoderm. Thus, in addition to brk and sog, other ventrally localized factors provide patterning information for the ectoderm. Candidates for this function are members of the spitz group of genes (Jazwinska, 1999b).

In the wing disc, brk mutant clones have completely cell autonomous effects that lead to cell fate transformations corresponding to low or intermediate levels of Dpp signaling (Campbell, 1999; Jazwinska, 1999a, and Minami, 1999). Mutant cells autonomously express the Dpp target genes optomotor blind (omb) and spalt (sal) without expressing dpp. omb and sal each represent a target gene of a different class with regard to brk regulation. While omb shows normal levels of expression in brk clones, sal, the target gene that depends on higher Dpp levels, is only weakly expressed. This indicates that sal integrates brk-dependent and brk-independent mechanisms for its activation. High-level target genes that are not affected by brk have not been found in wing discs so far; however, the cell fate transformation caused by ectopic expression of constitutively active Tkv (Tkv*) indicate that such target genes exist (Jazwinska et al., 1999). Since in the complete absence of Mad, which is essential for all aspects of Dpp signaling, removal of brk leads to the ectopic expression of omb and sal, it is concluded that Dpp signaling acts to relieve brk repression. brk is expressed in lateral regions of the wing disc in a pattern which might largely result from transcriptional repression by Dpp. This might also be the predominant mechanism by which Dpp signaling relieves Brk’s repression of target genes (Jazwinska, 1999b).

The relationship between dpp and brk is more complex in the embryo than in the wing imaginal disc for several reasons: (1) brk influences dpp expression in the embryo and thus changes the Dpp activity gradient. Therefore, it is more difficult to judge whether target gene mis-expression in brk mutants is a direct result of removal of brk or of the expansion of Dpp. (2) Three of the genes, dpp, zen and tld, whose expression is expanded in brk, are also targets of the maternal Dl gradient, and so are subject to both Dl-mediated and Brk-mediated repression in early embryos. (3) brk itself is clearly an activated target of Dl when it is first required for repression of dpp, tld and zen; only later during gastrulation does Dpp negatively regulate brk. (4) In the embryo but not in the disc, brk acts with sog in an intricate way to shape the Dpp gradient (Jazwinska, 1999b).

Despite these complexities brk’s relationship to the Dpp activity gradient in early embryos and to the Dpp gradient in wing discs is very similar. In both contexts low and intermediate-level targets are misexpressed in Brk mutants independent of Dpp signaling. Using brk;dpp double mutant embryos it has been demonstrated that the ectopic expression of zen and pnr in brk embryos is not a secondary consequence of the activation of dpp, but occurs even in the complete absence of Dpp signaling. pnr activation in brk;dpp double mutant embryos furthermore confirms that repression by brk is not dependent on prior repression by maternal Dl since pnr is not a direct target gene of Dl repression. pnr’s regulation by brk is strikingly similar to brk’s regulation of sal in the wing disc. pnr and sal both are intermediate-level Dpp target genes. In brk mutant cells, they both show weak levels of expression as compared with the expression in their normal domains. Thus, in the embryo and in the disc three types of Dpp target genes can be distinguished with regard to regulation by brk: (1) those that are fully activated upon loss of brk; (2) those that are weakly activated, and (3) those that are not affected. The regulation of the first group might occur entirely by antagonizing brk’s repression; the second group requires both relief of repression by brk and positive activation by Dpp; and the third group might only be subject to direct activation by Dpp (Jazwinska, 1999b).

brk-mediated target gene regulation might be important for morphogen function of Dpp. The strongest phenotypic effects of brk mutations seen so far are in places where Dpp acts as a morphogen. In both the wing disc and embryo brk regulates Dpp target genes that are activated at the lower end of the gradient. In these regions special mechanisms of gradient interpretation might be required that are sensitive to small changes in Dpp activity. Target gene regulation by brk could provide such a mechanism. The full activation of low-level target genes requires only transcriptional repression of brk by Dpp. However, a more non-linear mechanism suited to establish sharp threshold responses would result if target gene promoters had both repressive brk and activating SMAD binding sites. Then, Dpp would simultaneously downregulate brk transcription and antagonize its function at the target gene promoters. Such a dual mechanism is the more likely because the data demonstrate that it operates for the intermediate targets sal and pnr. However, experimental support for such a model can only come from a detailed analysis of how brk interacts with the target gene promoters (Jazwinska, 1999b).

PROTEIN STRUCTURE

Amino Acids - 704

Structural Domains

The protein sequence of Brk contains several regions of repeated amino acids. There are three glutamine-rich regions, an alanine- and histidine-rich region, a serine-rich region, and one run of eight histidines. Blast database searches that filter out these repeats fail to identify significant homology to any protein in the data banks. However, searching specifically with the N-terminal 100 amino acids of Brk reveals a weak homology between amino acids 44-99 and the homeobox domains from several proteins (21%-23% identities). The predicted secondary structure of this region of Brk contains two alpha helices, one from residues 10-16 and another from residues 31-56, and a less structured region in between them. Noticeably, the conserved amino acids (R-43, Q-44, W-48, Q-50), which are part of the DNA recognition helix in the homeodomain, are also present in the C-terminal alpha helix in this region of Brk. Although speculative, this suggests that Brk may bind to DNA. The sequence PMDLSLG at 377-383 is similar to a motif (P-DLS-K) present in several proteins known to act as transcriptional repressors that have been shown to interact with the corepressor CtBP (Nibu, 1998). In addition, there is a short stretch of basic amino acids (KKQRRLKKK) at position 517-525 that is reminiscent of the nuclear localization signal of SV40 large T antigen. The presence of these basic residues and the putative DNA-binding domain and transcriptional repression domain, together with the genetic data that Brk inhibits the pathway downstream of the SMADs, suggests that Brk functions as a repressor of Dpp target genes (Jazwinska, 1999a).

The most striking features of the amino acid sequence are homopolymer stretches encoded by opa (CAX) repeats; such repeats have no clear function. A search of databases for similarity to the nonrepetitive regions of Brk failed to identify any potential homologs or any regions of strong similarity to any regions of other proteins. As a first attempt to determine the possible function of this protein, its cellular localization was determined by immunofluorescence using a specific antibody to the protein. This shows that Brk protein expression in the wing disc is indistinguishable from that of the RNA and that the protein is localized to the nucleus. The sequence contains three potential nuclear localization signals. Although no obvious homologs have been identified by standard similarity searches, potential functional domains can be predicted by other means, including a method to identify potential helix-turn-helix (HTH) DNA-binding motifs. This procedure predicts Brk protein to have an HTH motif in the N-terminal region at positions 68-89. Another feature of the primary sequence is a central domain of about 150 amino acids, almost half of which are charged. Several repressor domains in transcription factors are characterized by having a similar high percentage of charged residues, suggesting this domain in Brk may play a similar role (Campbell, 1999).

date revised: 6 August 1999

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