u-shaped

Transcriptional Regulation

The dorsal ectoderm of the Drosophila embryo is subdivided into different cell types by an activity gradient of two TGFbeta signaling molecules, Decapentaplegic and Screw. Patterning responses to this gradient depend on a secreted inhibitor, Short gastrulation and a newly identified transcriptional repressor, Brinker, which are expressed in neurogenic regions that abut the dorsal ectoderm. The expression of a number of Dpp target genes has been examined in transgenic embryos that contain ectopic stripes of Dpp, Sog and Brk expression. These studies suggest that the Dpp/Scw activity gradient directly specifies at least three distinct thresholds of gene expression in the dorsal ectoderm of gastrulating embryos. Brk was found to repress two target genes, tailup/islet (tup) and pannier, that exhibit different limits of expression within the dorsal ectoderm. These results suggest that the Sog inhibitor and Brk repressor work in concert to establish sharp dorsolateral limits of gene expression. Evidence is provided that the activation of Dpp/Scw target genes depends on the Drosophila homolog of the CBP histone acetyltransferase (Ashe, 2000).

The dpp stripe results in an expansion in both the hnt and ush expression patterns. The broadening of these patterns is particularly evident in anterior regions in the vicinity of the eve stripe. Increases in dpp+ gene dose do not expand the pnr expression pattern. For example, four copies of dpp+ result in augmented levels of pnr expression, but the dorsoventral limits of expression are essentially normal. The stripe2-dpp transgene has no obvious effect on the early sog and brk expression patterns (Ashe, 2000).

Previous studies have identified mutations in the Drosophila homolog of the mammalian CBP histone acetyltransferase gene, nejire. nej is maternally expressed so that the detection of early patterning defects depends on the analysis of embryos derived from females containing nej germline clones. The complete loss of nej+ activity results in a failure to make mature eggs. However, it is possible to obtain embryos from a strong hypomorphic allele, nej¹. These embryos exhibit dorsoventral patterning defects. Recent studies have shown that CBP interacts with Smad proteins including the Drosophila protein Mad, a transcription factor downstream of Dpp signaling. In nej mutant embryos, there is a loss of the amnioserosa and other derivatives of the dorsal ectoderm. The expression of target genes requiring peak levels of Dpp signaling is essentially abolished. For example, hnt expression is lost in the presumptive amnioserosa, but persists at the posterior pole where it might be separately regulated by the torso signaling pathway (Ashe, 2000).

There is a similar loss of the dorsal rho pattern in mutant embryos. In contrast, the lateral, neurogenic stripes are unaffected, indicating that the nej mutant does not cause defects in the patterning of the neurogenic ectoderm. Moreover, the fact that the rho stripes are excluded from ventral regions, as seen in wild-type embryos, suggests that the patterning of the mesoderm is also normal. Thus, the nej mutation does not appear to cause a general loss of transcriptional activation, but instead results in specific patterning defects in the dorsal ectoderm. Target genes that are activated by lower levels of Dpp signaling such as ush and pnr are also affected by the nej mutation. In the case of ush, there is a loss of staining in central regions of the dorsal ectoderm. Moreover, the residual staining pattern is narrower than the wild-type pattern. This is reminiscent of the ush pattern seen in dpp/+ heterozygotes. However, the nej mutation also causes a narrowing of the pnr pattern, whereas expression is normal in dpp/+ embryos (Ashe, 2000).

A summary is presented of Dpp signaling thresholds in the embryo. The Dpp/Scw activity gradient presumably leads to a broad nuclear gradient of Mad and Medea across the dorsal ectoderm of early embryos. It is conceivable that the early lateral stripes of brk expression lead to the formation of an opposing Brk repressor gradient through the limited diffusion of the protein in the precellular embryo. Peak levels of Dpp and Scw activity lead to the activation of Race and hnt at the dorsal midline. The tup and ush patterns represent another threshold of gene activity. The similar patterns might involve different mechanisms of Dpp signaling since tup is repressed by Brk, whereas ush is not. Finally, the broad pnr pattern represents another threshold of gene activity. It is not inhibited by Sog but is repressed by Brk. It is possible that tup and pnr are differentially repressed by a Brk gradient. Low levels of Brk might be sufficient to direct the lateral limits of the tup pattern, whereas high levels may be required to repress pnr (Ashe, 2000).

The morphogen gradient of Wingless provides positional information to cells in Drosophila imaginal discs. Elucidating the mechanism that precisely restricts the expression domain of wingless is important in understanding the two-dimensional patterning by secreted proteins in imaginal discs. In the pouch region of the wing disc, wingless is induced at the dorsal/ventral compartment boundary by Notch signaling in a compartment-dependent manner. In the notum region of the wing disc, wingless is also expressed across the dorsal/ventral axis, however, regulation of notal wingless expression is not fully understood. Notal wingless expression is established through the function of Pannier, U-shaped and Wingless signaling itself. Initial wingless induction is regulated by two transcription factors, Pannier and U-shaped. At a later stage, wingless expression expands ventrally from the pannier expression domain by a Wingless signaling-dependent mechanism. Interestingly, expression of pannier and u-shaped is regulated by Decapentaplegic signaling that provides the positional information along the anterior/posterior axis, in a concentration-dependent manner. This suggests that the Decapentaplegic morphogen gradient is utilized not only for anterior/posterior patterning but also contributes to dorsal/ventral patterning through the induction of pannier, u-shaped and wingless during Drosophila notum development (Tomoyasu, 2000).

A hierarchy of the activity of these genes during notum development is presented. dpp is initially induced at the dorsal region of the A/P compartment boundary by Hh signaling. Dpp signaling induces two target genes, pnr and ush. Analyses of pnr expression in put-ts and tkv^a12 cells suggest that different thresholds are set for the induction of these genes: low levels for pnr and high levels for ush. wg is induced by Pnr where ush is not expressed. Simultaneously, the Pnr-Ush complex represses wg expression at the dorsal-most region of the presumptive notum. In the later stage, the wg expression domain expands ventrally from the pnr expressing region and wg starts to be expressed in the non-pnr-expressing cells. During this process, Wg signaling plays a crucial role and this separation does not occur in the Wg signaling mutants. The Pnr-Ush complex acts as a repressor for the induction of wg and of DC enhancer-lacZ expression (DC enhancer is an enhancer of the achaete-scute proneural gene complex that activates gene expression in the dorsocentral area). It is interesting that Ush does not simply inhibit Pnr function but switches the activator function of Pnr to a repressor function. Based on the result that the extra doses of Pnr cannot revert the repressor activity of Pnr-Ush, it has been proposed that the activator function of Pnr and the repressor function of the Pnr-Ush complex do not simply compete with each other on the notal wg enhancer element. However, it also seems to be possible that Pnr and the Pnr-Ush complex compete for the binding site at the notal wg enhancer, but the ability of Pnr-Ush complex to bind this site may be greater than that of Pnr. It is also worth noting that FOG-1, a mammalian homolog of Ush, represses the transactivation of alpha-globin and EKLF promoter by GATA-1, but enhances the transactivation of NF-E2 p45 promoter by GATA-1 in a culture cell system. Dorsocentral (DC) bristles are ectopically formed but postvertical bristles on the head are missing in a loss-of-function allelic combination for ush or in pnr^D1 heterozygous flies. These observations suggest that the Pnr-Ush complex acts as a repressor for the DC enhancer, but acts as an activator for the enhancer of postvertical bristles. Only a cis-regulatory element of the DC enhancer has been analyzed at the nucleotide level. Additional studies of the molecular analyses of the cis-regulatory elements of both wg and DC or other enhancers of the achaete-scute complex seem to be necessary in order to reveal the functions of Pnr and Ush (Tomoyasu, 2000).

Generally, at least two different coordinate axes are necessary for positional specification in a two-dimensional field. Morphogen gradients of Dpp and Wg provide this axial information during Drosophila imaginal disc development. In both wing and leg discs, Dpp is induced at the A/P compartment boundary by Hh signaling. In the leg disc, wg is also induced by Hh signaling. Mutual repression between Dpp and Wg signaling separates each expression territory, localizing dpp in the dorsal and wg in the ventral regions abutting the A/P border (a compartment-independent manner). In contrast, wg is induced by Notch signaling only at the D/V compartment boundary in the wing pouch (a compartment-dependent manner). Then, secreted Dpp and Wg proteins provide positional information along the A/P and D/V axes, respectively, to establish Cartesian-like coordinates in the pouch field. Relative positions of dpp and wg expression domains in the notum are more similar to those in the wing pouch (in both cases, the expression domains are orthogonal). However, a D/V compartment boundary does not exist in the notum. The results described here reveal that another compartment-independent mechanism acts to pattern the presumptive notum. Namely, the D/V axis, provided by Pnr, Ush, and Wg, is initially established by the Dpp gradient, which mainly contributes the positional information along the A/P axis. One of the key issues of this patterning model is that Dpp signaling seems to act preferentially along the A/P axis of the notum. This is because two target genes, pnr and ush, are induced farther from the Dpp source along the A/P axis than along the D/V axis. One possible explanation for this phenomenon is that the diffusion of Dpp protein may be positively regulated along the A/P axis. However, such asymmetric induction is not observed on the dad induction; dad is one of the Dpp signaling targets in the wing disc. This suggests that diffusion of Dpp protein is not directionally regulated in the notum region. An alternative explanation would be that an effective range of Dpp morphogen gradient is established in a relatively short range. Cells that respond to Dpp would proliferate or migrate preferentially along the A/P axis. pnr mRNA is detected mainly in the posterior-dorsal region of the presumptive notum. GFP expression of UAS-gfp pnr^md237 is seen along the entire dorsal side of the presumptive notum. This difference between the staining pattern of pnr mRNA and GFP expression of UAS-gfp pnr^md237 in the late third larval stage seems to be caused by a long half-life of gal4 and/or gfp products, suggesting that cells that once have expressed pnr mRNA proliferate preferentially along the A/P axis. However, it seems to be difficult to explain the determination of pnr and ush expression domains only by the Dpp morphogen gradient. The existence of Tkv*-insensitive cells for inducing pnr and ush indicates that some regional subdivision may occur independently of Dpp signaling. Discontinuous expression of dpp in the A/P border of the notum also suggests the existence of a Dpp-independent subdivision. D/V subdivision of the presumptive notum seems to be achieved by several parallel mechanisms, including Dpp signaling (Tomoyasu, 2000).

Because Drosophila is a holometabolous insect, it should destroy larval tissues and replace them with a different population of cells to form the adult structure during the pupal stage. Thus, formation of epidermal structure should occur reiteratively during embryogenesis and metamorphosis. Patterning of larval epidermal structure takes place during embryogenesis; however, patterning of adult structure is mainly performed in larval stage imaginal discs. The Dpp morphogen gradient has been shown here to regulate pnr and ush expression to pattern the presumptive notum, which forms the dorsal structure of the adult, in the wing imaginal disc. pnr and ush are necessary for the formation of amnioserosa, the dorsal structure of the embryo, and both pnr and ush expressions are also positively regulated by Dpp in a concentration-dependent manner during embryogenesis. Furthermore, dorsal closure during embryogenesis and thorax closure in metamorphosis is also analogous, because both processes are regulated by the same signaling cascade, JNK signaling. These similarities between embryogenesis and metamorphosis presumably reflect the evolutionary history of the development in holometabolous insects (Tomoyasu, 2000).

Targets of Activity

The pattern of the large sensory bristles on the notum of Drosophila arises as a consequence of the expression of the achaete and scute genes. U-shaped acts as a transregulator of achaete and scute in the dorsal region of the notum. Viable hypomorphic u-shaped mutants display additional dorsocentral and scutellar bristles that result from overexpression of achaete and scute. A synergism between ac-sc and ush has been observed: animals heterozygous for both a deletion of ush and a deletion of the Achaete-Scute complex lack the posterior vertical bristles on the head. ac-sc mutants are epistatic over ush for the bristle phenotype: in the absence of ac-sc function, no sensory organs develop, even in ush flies. This indicates that Ush functions upstream of Ac and Sc. The additional macrochaetes seen in ush mutants may therefore be attributable to overexpression of ac and sc. Overexpression of u-shaped causes a loss of achaete-scute expression and consequently a loss of dorsocentral bristles (Cubadda, 1997).

The effects of u-shaped on the dorsocentral bristles appear to be mediated through the enhancer sequences that regulate achaete and scute at this site. The dorsocentral proneural cluster of ac-sc expression is known to depend on enhancer sequences, as for example, the dorso-central (DC) enhancer, located 4.0-9.8 kb upstream of the ac start site (Cubadda, 1997). The effects on u-shaped mutants are similar to those of a class of dominant alleles of the gene pannier with which they display allele-specific interactions, suggesting that the products of both genes cooperate in the regulation of achaete and scute. A study of the sites at which the dorsocentral bristles arise in mosaic u-shaped nota, suggests that the levels of the u-shaped protein are crucial for the precise positioning of the precursors of these bristles (Cubadda, 1997).

On each half of the dorsal mesothorax (heminotum), 11 large bristles (macrochaetae) occupy precisely constant positions. The location of each macrochaeta is specified during the third instar larval and early pupal stages by the emergence of its precursor cell (sensory mother cell: SMC) at a precise position in the imaginal wing discs, the precursors of the epidermis of most of the mesothorax and wings. The accurate positioning of SMCs is thought to be the culmination of a multistep process in which positional information is gradually refined. The GATA family transcription factor Pannier and the Wnt secreted protein Wingless are known to be important for the patterning of the notum. Thus, both proteins are necessary for the development of the dorsocentral mechanosensory bristles. Pannier has been shown to directly activate the proneural genes achaete and scute by binding to the enhancer responsible for the expression of these genes in the dorsocentral proneural cluster. Moreover, the boundary of the expression domain of Pannier appears to delimit the proneural cluster laterally, while antagonism of Pannier function by U-shaped, a Zn-finger protein, sets its limit dorsally. Therefore, Pannier and U-shaped provide positional information for the patterning of the dorsocentral cluster. In contrast and contrary to previous suggestions, Wingless does not play a similar role, since the levels and vectorial orientation of its concentration gradient in the dorsocentral area can be greatly modified without affecting the position of the dorsocentral cluster. Thus, Wingless has only a permissive role on dorsocentral achaete-scute expression. Evidence is provided indicating that Pannier and U-shaped are main effectors of the regulation of wingless expression in the presumptive notum (Garcia-Garcia, 1999).

An enhancer that directs expression specifically at the DC proneural cluster is present within a 5.7 kb fragment of AS-C DNA. Different subfragments were assayed for enhancer activity in vivo. A 1.4 kb subfragment (AS1.4DC) directs lacZ transcription from a minimal hsp70 promoter in the DC proneural cluster: beta-galactosidase and Scute endogenous accumulations precisely colocalized at this cluster. This fragment and the corresponding region of the AS-C from D. virilis were sequenced. Stretches of conserved DNA were present throughout the fragment, although they appeared to cluster within three regions. Subfragments containing each one of these regions were assayed for DC enhancer activity. Only the most 3' subfragment (PB0.5DC) shows such an activity, but to a much lesser extent than AS1.4DC. Interestingly, the activity is usually limited to only one cell, which is the posterior DC SMC. However, when assayed with the sc promoter, the PB0.5DC fragment directs lacZ activity in most cells of the DC cluster. Consequently, the sequences essential for specifying transcription in the DC cluster are contained within the PB0.5DC subfragment, although additional sequences that reinforce this expression are present in the larger AS1.4DC fragment. The AS1.4DC fragment was used to study DC enhancer activity (Garcia-Garcia, 1999).

The Pnr protein is known to regulate ac-sc expression at the DC cluster by acting directly or indirectly through the DC enhancer. The sequence of AS1.4DC was examined: within it, seven putative GATA-1 factor binding sites were found. Three of them fit the vertebrate consensus sequence (WGATAR: sites 1, 2 and 4); three comply with the consensus obtained in a random oligonucleotide selection experiment performed with Pnr protein (GATAAG: sites 3, 5 and 6), and one fits both consensus sequences (site 7). In the prospective notum, the stripe of diffusible Wg protein straddles the lateral border of the domain of expression of pnr. This is compatible with the location of the Wg source being on the border of, but still within, the pnr domain. In accordance with this location, pnr appears to activate wg, since it has been found that a wg-lacZ construct, which reproduces the notal band of Wg accumulation, is not expressed in pnr mutant discs and is ectopically expressed in the dorsalmost area of the disc in a pnr dominant gain-of-function combination. In contrast, other data suggest that Pnr represses wg. Thus, the notal wg stripe is expanded dorsally in strong hypomorphic pnr combinations. Moreover, in flies in which pnr is overexpressed there was no expansion of the domain of WG mRNA, which in fact accumulates in a stripe that is even narrower than that seen in the wild type. The repressing effects appeared to be restricted to the domain of accumulation of Ush, which suggests the participation of Pnr/Ush heterodimers in the repression. Consistent with this assumption, the Pnr^D1 mutant protein, which is incapable of interacting with Ush, promotes wg expression within the entire dorsalmost area of the disc in pnr mutants animals. Interestingly, Pnr D1 can not induce the expansion of the wg expression domain in the presence of wild-type Pnr, suggesting that Pnr+/Ush heterodimers interfere with the Ush-resistant function of Pnr^D1. Such interference may also account for the repression of the Pnr^D1-mediated dorsal expansion of DC-lacZ expression by Pnr+. Taken together, these results suggest that during development of the wing disc, Pnr is necessary both for activation of wg and (together with Ush) for its repression in the dorsalmost region of the presumptive notum. This dorsal repression probably takes place from the start of wg expression, since the earliest detectable accumulation of WG mRNA is already restricted to the presumptive mid notal region. A wg-lacZ enhancer trap line, which shows expression throughout the dorsalmost part of the early third instar wing discs and posterior refinement to the notal stripe, might have a reduced sensitivity to the repression by Pnr/Ush (Garcia-Garcia, 1999).

A model is provided for the dorsal-lateral patterning of the DC area by Pnr and Ush. In the third instar wing disc and in the dorsalmost part of the prospective notum, Ush is present at high concentrations and the Pnr/Ush heterodimers are relatively abundant. These heterodimers would act as repressors and prevent activation of downstream genes. In the DC area, defined along the dorso-lateral axis by lower concentrations of Ush and the presence of Pnr, there is sufficient free Pnr to activate genes like ac-sc, DC-lacZ and wg. ac-sc is transcribed in the more dorsal part of the area because its activation requires relatively high concentrations of Pnr. wg is only transcribed at the edge of the Pnr domain because its expression is very sensitive to both Pnr and Pnr/Ush, and consequently low concentrations of the former are sufficient for activation and low concentrations of the latter, even in the presence of high concentrations of free Pnr, impose repression. The inability of extra doses of the activator Pnr to revert the repression by Pnr/Ush in the dorsalmost region of the notum suggests that activator and repressor do not compete for overlapping sites at the DC as-sc and notal wg enhancers. The presence of Pnr/Ush at their site(s) would block the activating effect of bound Pnr. Additional inputs, notably decapentaplegic, are known to act on the DC enhancer (Garcia-Garcia, 1999).

In Drosophila, muscles attach to epidermal tendon cells are specified by the gene stripe (sr). Flight muscle attachment sites are prefigured on the wing imaginal disc by sr expression in discrete domains. The mechanisms underlying the specification of these domains of sr expression have been examined. The concerted activities of the wingless (wg), decapentaplegic (dpp) and Notch (N) signaling pathways, and the prepattern genes pannier (pnr) and u-shaped (ush) establish domains of sr expression. N is required for initiation of sr expression. pnr is a positive regulator of sr, and is inhibited by ush in this function. The Wg signal differentially influences the formation of different sr domains. These results identify the multiple regulatory elements involved in the positioning of Drosophila flight muscle attachment sites (Ghazi, 2003).

Pnr, a GATA-binding protein normally functions as a transcriptional activator and is antagonized by Ush in its function. Loss of function pnr mutants show no sr expression in the domain covered by pnr. This, along with sr expansion in mutants of ush, would suggest that pnr activates sr in the notum, and is inhibited by ush. However, there is also loss of sr expression in pnr `gain of function' mutants. The reason for this is not completely clear. One possibility is that since the mutation causes an increase in wg activity in the region this may cause a down-regulation of sr. This is supported by a similar effect seen on misexpression of activated armadillo in the pnr domain. Results with both pnr and ush have been taken into account to suggest that pnr positively regulates sr and is antagonized by ush (Ghazi, 2003).

These results indicate that each sr domain is regulated by a combination of prepattern genes and signaling molecules. But, a precise description of the 'combinatorial code' for regulation of each sr domain is beyond the scope of this work and can be achieved by generation of domain specific markers for sr. Based on expression pattern data, and existing literature, it is suggested that high levels of Pnr, low (or absence of) Ush and moderate levels of Wg determine the initial induction of domain a. The distinction between medial (a) and lateral (b-d) domains is established by the presence of very high levels of Wg (the cells where the Wg gradient originates). Lateral expression domains are probably induced in domains controlled by the lateral prepattern gene iro. The differences between different lateral domains arise as a result of expression of different genes in the region. For instance, the lateral-most domain d appears to be regulated by ush and does not encounter Wg at all. Whereas, all cells of b receive uniformly moderate levels of Wg, only cells at the borders of c receive high Wg levels, and these differences result in the distinct identities of the two domains. Dpp, either through its effects on these regulatory genes and/or through direct effects on sr influences the process (Ghazi, 2003).

Protein Interactions

The genes pannier (pnr) and u-shaped (ush) are required for the regulation of achaete-scute during establishment of the bristle pattern in Drosophila. pnr encodes a protein belonging to the GATA family of transcription factors, whereas ush encodes a novel zinc finger protein. Genetic interactions between dominant pnr mutants bearing lesions situated in the amino-terminal zinc finger of the GATA domain and ush mutants have been described. The number of ectopic bristles in pannierD/+ flies increases in flies bearing only a single copy of u-shaped+ but decreases when three copies are present. Activation of a chicken alpha-globin promoter sequence by Pannier in cultured cells is inhibited by Ush. When both Ush and wild-type Pnr are expressed simultaneously, activation is abolished. Stimulation by Pnr is lost progressively in a concentration-dependent manner. Similarly, activation by chicken GATA-1 is also lost after cotransfection with the Ush expression vector. Because Pnr and GATA-1 have no homology outside their GATA DNA-binding domain, and since Ush alone has no effect on globin promoter activity, these observations suggest that the function of Ush is mediated through the GATA DNA-binding domain (Haenlin, 1997).

Additional genetic evidence is provided for an antagonistic interaction of Pannier and U-shaped. Pnr proteins deleted in the C-terminal region do not activate transcription from a heterologous alpha-globin promoter. Overexpression of wild-type and mutant Pnr proteins in transgenic flies regulates achaete-scute expression through the dorsocentral enhancer. Heterozygous flies mutant for pannierD alleles differentiate extra dorsocentral bristles resulting from overexpression of achaete-scute, whereas heterozygous flies mutant for C-terminally deleted Pnr proteins differentiate fewer dorsocentral bristles attributable to decreased ac-sc expression. Overexpression of the U-shaped protein in transgenic flies reduces achaete-scute expression in wild-type but not in pannierD mutants. It is concluded that Ush antagonizes the effects of Pnr, leading in consequence to reduced achaete-scute expression and reduced bristle development (Haenlin, 1997).

Pnr and Ush are found to heterodimerize through the amino-terminal zinc finger of Pnr; when associated with Ush, the transcriptional activity of Pnr is lost. In contrast, the mutant pnr protein with lesions in this finger associates only poorly with Ush and activates transcription even when cotransfected with Ush. The results suggest an antagonistic effect of Ush on Pnr function and reveal a new mode of regulation of GATA factors during development (Haenlin, 1997).

serpent encodes a GATA transcription factor essential for hematopoiesis in Drosophila. Previously, Srp was shown to contain a single GATA zinc finger of C-terminal type. srp encodes different isoforms, generated by alternative splicing, that contain either only a C-finger (SrpC) or both a C- and an N-finger (SrpNC). The presence of the N-finger stabilizes the interaction of Srp with palindromic GATA sites and allows interaction with the Friend of GATA factor U-shaped (Ush). The respective functions of SrpC and SrpNC during embryonic hematopoiesis were examined. Both isoforms individually rescue blood cell formation, which is lacking in a srp null mutation. Interestingly, while SrpC and SrpNC activate some genes in a similar manner, they regulate others differently. Interaction between SrpNC and Ush is responsible for some but not all aspects of the distinct activities of SrpC and SrpNC. These results suggest that the inclusion or exclusion of the N-finger in the naturally occurring isoforms of Srp can provide an effective means of extending the versatility of srp function during development (Waltzer, 2002).

In a systematic search for GATA zinc finger-coding sequences in the Drosophila genome, five genes were found: dGATA-E (CG10278), dGATA-D (CG5034), pnr, grain and srp. dGATA-E and dGATA-D appear to include only a C-finger, while Pnr and Grain contain both an N- and a C-finger. Interestingly, while Srp has been reported to contain a single C-finger, the presence of a putative exon (E4A) coding for an N-finger motif in srp is also evident. Using RT–PCR assays with various combinations of oligonucleotides, it has been shown that E4A is expressed and that E4A and E4B are alternatively spliced to exon 5. In the course of these experiments, an additional splice acceptor site was also identified within E7. This downstream acceptor site in E7 is out-of-frame and leads to the deletion of the Srp glutamine-rich C-terminal region. The data indicate that four alternatively spliced mRNAs are transcribed from srp, two encoding products with a single C-finger (SrpC and SrpCd) and two encoding products with both N- and C-fingers (SrpNC and SrpNCd). Interestingly, in SrpNC and SrpNCd, the two fingers present the same conserved organization as in other GATA factors. Notably, they are separated by 29 amino acids, as in all vertebrate GATA. The two isoforms that contain the full-length exon 7, i.e. srpC and srpNC, have been used to address the functional consequences of the alternative splicing of E4A and E4B (Waltzer, 2002).

Whether SrpC and SrpNC display different properties in vitro was investigated. While the C-finger is necessary and sufficient for specific DNA binding, it has also been shown in vertebrates that the N-finger can stabilize the binding to particular double GATA sites. By electophoretic mobility shift assays (EMSAs), it was determined if SrpNC and SrpC have similar DNA-binding properties. Both in vitro translated SrpC and SrpNC proteins bind to an oligonucleotide containing a consensus GATA site. The binding is specific, since it can be competed out efficiently by an excess of cold GATA oligonucleotide, but not by an excess of the GATC oligonucleotide. The stability of the SrpN and SrpNC complex on a single or on a palindromic GATA site was assessed by dissociation experiments. While the rate of dissociation is similar for SrpC and SrpNC on a single GATA probe, SrpNC bound more stably than SrpC to the palindromic GATA sites (Waltzer, 2002).

The GATA N-finger allows interaction with cofactors of the FOG family. Key residues that are required for the interaction between GATA and FOG are conserved in the Srp N-finger. In order to test the binding between Ush and srp products, pull-down assays were performed in vitro. in vitro translated [³⁵S]methionine-labelled Ush binds to GST–SrpNC, but not GST–SrpC. Thus, Ush specifically interacts with Srp isoforms that contain the N-finger. In addition, like its vertebrate homologs, Ush interacted with the transcriptional corepressor dCtBP in this assay (Waltzer, 2002).

Taken together, the results indicate that SrpNC displays features characteristic of two-fingered GATA factors. The two types of naturally occurring isoforms encoded by srp (with or without the N-finger) have different DNA-binding properties, and only the isoforms including an N-finger can interact with Ush (Waltzer, 2002).

In order to determine whether a spatial regulation of the alternative splicing leading to SrpC and SrpNC occurs during embryonic development, the distribution of the corresponding srp transcripts was assessed by in situ hybridization using specific probes for exon 4A or 4B. At the blastoderm stage and during gastrulation, srpC and srpNC show the same expression pattern. They are expressed in the procephalic mesoderm, the hemocyte primordium, at the anterior and posterior pole, in the primordium of the anterior and posterior midgut as well as in the amnioserosa and in the yolk cells. Later, during germ band extension, and after germ band retraction, srpC and srpNC are expressed identically in the developing fat body. Thus, srpC and srpNC transcripts are not differentially regulated spatially during embryonic development. However, the level of the transcripts is not identical. Indeed, by means of semi-quantitative RT–PCR, it was determined that exon 4B-containing mRNA is five times more abundant than exon 4A-containing mRNA, suggesting that two-fingered isoforms of Srp are less abundant than single-fingered isoforms (Waltzer, 2002).

In order to analyse SrpC and SrpNC activities, their capacities to activate gene expression in vivo were tested during Drosophila embryonic hematopoiesis. Using the UAS-GAL4 system, they were ectopically expressed in the mesoderm and the expression pattern of various hematopoietic markers was assessed. The two genes ush and gcm play critical roles in embryonic hematopoiesis. Their expression in the hematopoietic primordium occurs early and appears to depend on srp activity. Therefore, it was determined whether they are transcriptional targets of SrpC and/or SrpNC. Whereas in a wild-type early embryo, ush expression is restricted to the anterior mesoderm, twist-driven expression of SrpC (twist-SrpC) or SrpNC (twist-SrpNC) induces strong expression of ush throughout the mesoderm. In contrast, twist-SrpC induces gcm expression poorly and in a limited number of mesodermal cells of stage 5 embryos, whereas twist-SrpNC strongly activates gcm expression segmentally from stage 5 to 9 (Waltzer, 2002).

The expression of hematopoietic lineage-specific markers was examined. As plasmatocyte markers, peroxidasin (pxn) and croquemort (crq) were used. Since, crystal cells are the only source of prophenoloxidase (pro-PO) in Drosophila, expression of this gene was used to monitor crystal cell formation. pro-PO transcripts were indeed detected in these cells from early stage 11 to the end of embryogenesis. Analysing these markers, two situations were observed. twist-SrpC and twist- SrpNC have similar abilities to induce expression of the plasmatocyte marker pxn and of the crystal cell marker pro-PO, however expression of crq was induced by twist-SrpC and not by twist-SrpNC. Note that pxn and crq were induced through most of the mesoderm, while pro-PO activation was restricted to the head region (Waltzer, 2002). Taken together, these data show that SrpC and SrpNC have both common and different activities during hematopoiesis. Indeed, both isoforms activate the expression of ush, pxn and pro-PO in a similar manner. However, SrpC and SrpNC differentially stimulate the expression of crq and gcm, respectively, in the mesoderm (Waltzer, 2002).

It is remarkable that srp encodes both single and dual zinc finger-containing products. The results provide strong evidence that this alternative splicing allows production of transcription factors with specific activities. The two isoforms activate the expression of ush and pxn with similar efficiency, suggesting that SrpC and SrpNC have similar transactivating properties in vivo, yet, SrpC (but not SrpNC) activates crq expression, while SrpNC is a much stronger activator of gcm expression than SrpC. The domain coded by exon 4B that is present only in SrpC has no known motif and it is not known if and how it participates in SrpC-specific function. However, the presence of the N-terminal zinc finger encoded by exon 4A may explain some of the distinct features of SrpNC as discussed below (Waltzer, 2002).

As in the case of vertebrate GATA-1, the presence of the N-finger in Srp stabilizes binding to double palindromic GATA sites. Although the N-finger of GATA-1 modulates the binding and the transactivating properties of GATA-1 on synthetic promoters, the functional importance of these effects has remained elusive, particularly since no GATA-1 isoform contains only the C-finger. In the case of srp, these distinct binding properties may have direct functional consequences. For instance, the fact that SrpC and SrpNC activate a common target, ush, whereas only SrpNC strongly activates a specific target, gcm, could be related to the DNA-binding specificity of the two isoforms. A scan of the ush upstream regulatory region shows that it contains several GATA consensus sequences, nine of which are clustered in <1 kb and are organized as three repetitions of three sites. In contrast, GATA sites are far less frequent in gcm regulatory regions and are often organized in palindromes. Considering that ush and gcm are likely to be direct target genes for srp, the different organization of their regulatory regions may explain the differential effect observed (Waltzer, 2002).

The lack of plasmatocyte and crystal cell formation due to an srp null mutation can be rescued by expressing SrpC or SrpNC in the mesoderm. No difference between the two isoforms was seen in this assay, suggesting that the N-finger is not absolutely required for srp function in embryonic blood cell formation. However, in the absence of a functional test, to what extent the formation of embryonic blood cells is fully rescued cannot be determined. Interestingly, rescue experiments with the mouse GATA-1 mutant indicate that the GATA-1 N-finger is dispensable for primitive erythropoiesis but is required for definitive erythopoiesis. In Drosophila, a second wave of hematopoiesis, occurring at the larval stage, gives rise to four different lineages: plasmatocytes, crystal cells, secretory cells and lamellocytes. srp is expressed in the dorsal lymph gland (i.e. the main larval hematopoietic organ) and it probably controls larval hematopoiesis. By analogy to vertebrate GATA-1, the Srp N-finger may provide an additional function for larval hematopoiesis, perhaps during formation of the new cell types (Waltzer, 2002).

In the assay used, the expression of the transgene was limited to the mesoderm but it still rescued blood cell formation. This finding suggests that the early expression of srp in the hematopoietic primordium is sufficient to initiate the genetic program that controls hemocyte formation and differentiation. Interestingly, in the wild-type embryo, srp transcripts are not expressed detectably in hemocytes after stage 11, but Srp protein is detected in plasmatocytes and crystal cells throughout most of embryogenesis. Persistence of srp products in hemocytes might be critical for srp function, and control of srp products at the post-translational level may play a crucial role in the correct regulation of blood cell differentiation. Rescue of crystal cell formation by mesodermal expression of SrpC and SrpNC contrasts with the observation that later expression driven by lz-Gal4 in crystal cells represses their development. Srp levels are reduced in crystal cells compared with surrounding plasmatocytes. Therefore, the results are consistent with a two-step model in which Srp expression is first necessary to induce lz expression and subsequently is downregulated to allow crystal cell differentiation (Waltzer, 2002).

One of the best characterized features of GATA N-fingers is their dimerization with cofactors of the FOG family. Consistent with this feature, it was found that SrpNC interacts with the Drosophila FOG Ush, but SrpC does not. Previous analysis showed that ush regulates the number of crystal cells. It was proposed that this function of ush could be mediated by a putative isoform of Srp containing an N-finger. The current findings strongly support this hypothesis. However, it was not possible to address this issue directly, since both SrpC and SrpNC display a strong Ush-independent repressive effect on crystal cell formation and differentiation (Waltzer, 2002).

A new function of ush revealed here is the regulation of the level of expression of the macrophage receptor crq, suggesting that ush displays a broader function in hematopoiesis than previously assumed. Notably, evidence is provided that Ush modulates SrpNC transactivation of crq. Since Ush interacts with the corepressor dCtBP in vitro, the Ush–SrpNC complex could repress crq expression. However, it is not known whether crq is a direct target of srp, so the possibility that the Ush–SrpNC complex activates a transcriptional repressor that regulates crq cannot be ruled out. Vertebrate FOGs can act as either a coactivator or a corepressor of GATA factors. In Drosophila, Ush is a repressor of Pannier-induced activation in cell culture, and it probably also represses the expression of achaete in the dorso-central proneural cluster in vivo. Furthermore, in a heterologous assay in Drosophila, the CtBP-binding region of mFOG2 is required for repressing the formation of crystal cells but not cardiac cells. Thus several mechanisms seem to regulate the function of the GATA–FOG complex (Waltzer, 2002).

Remarkably, some functions of SrpNC appear to be independent of Ush. Thus, gcm-specific activation by SrpNC is not affected in an ush mutant embryo. Moreover, SrpNC still represses crystal cell formation in the absence of ush. This is reminiscent of mouse erythropoiesis, where both FOG-dependent and FOG-independent regulation of gene expression by GATA-1 have been observed. The molecular mechanisms underlying the regulation by Ush/FOG-1 of SrpNC/GATA-1 activity on some specific targets remain to be elucidated. It is tempting to speculate that the N-finger of SrpNC is involved in the recognition of promoter sequences, on gcm for example, and thus is not available to recruit Ush. Alternatively, other cofactors already localized to the promoter or bound to SrpNC might prevent Ush binding to the N-finger (Waltzer, 2002).

This study has focussed on hematopoiesis, but srp also participates in other developmental processes, such as germ band retraction, midgut differentiation, fat body formation, induction of the immune response and the ecdysone response. It will be interesting to determine the respective roles of SrpC and SrpNC in these different phenomena. Phylogenetic analysis shows that SrpNC is closely related to vertebrate GATA factors. It has been suggested that srp is a functional homolog of the entire vertebrate GATA family, since srp is required in Drosophila for hematopoiesis, like GATA-1/2/3 in mice, and for endodermal development, like GATA-4/5/6. Nevertheless, this hypothesis was at odds with the fact that Srp seemingly had a single zinc finger while all the vertebrate GATAs have two. The present identification of Srp isoforms with two fingers gives new force to this hypothesis. Further, the expression of isoforms of Srp with distinct activities helps to account for the broad range of functions ensured by this gene (Waltzer, 2002).

It is worth noting that alternative splicing eliminating the N-finger has also been described in Bombyx mori GATAß and in chicken GATA-5 genes. Moreover, a BLAST search analysis revealed alternatively spliced human expressed sequence tags coding for two isoforms of a potential GATA factor with either one or two zinc fingers. This suggests that alternative splicing of GATA genes could be more general than previously thought, and as yet unnoticed splice variants of GATA vertebrate genes may generate proteins with only a C-finger (Waltzer, 2002).

In conclusion, these results shed further light on the molecular control of hematopoiesis by the GATA factor Srp. The alternative splicing of srp gives rise to different Ush-interacting and non-interacting Srp proteins with different target gene specificities, thereby contributing to the exquisite control of Drosophila blood cell formation. It is speculated that alternative splicing of the GATA N-finger might be an important mechanism regulating the activity of other GATA genes from insects to man (Waltzer, 2002).

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