u-shaped

DEVELOPMENTAL BIOLOGY

Embryonic

See the embryonic expression pattern of ush at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

The 4.7 kb transcript is detected with a peak of expression during early embryonic stages at 4-8 hours (Cubadda, 1997).

Larval

Expression of ush in the late third-instar wing imaginal disc appears to be restricted to specific domains. Staining is detected in territories corresponding to the future dorsal-most region of the thorax, as well as in part of the hinge region and the posterior region of the pleura. In the hinge region, ush expression is found in a domain comprising the sites of appearance of the anterior notal wing process and the proximal tegula, expanding up to the border where anterior and posterior notopleural bristles develop. In the dorsal part of the notum, staining covers the site of appearance of the scutellar bristles and extends to the border of the site at which the dorsocentral bristles form. Therefore, in the notum, the area of ush expression corresponds well with the region where ush is required for normal development (Cubadda, 1997).

Effects of Mutation or Deletion

Germband retraction in Drosophila, like most embryonic morphogenetic events in this organism and in higher eukaryotes, is not well understood. Several approaches have been taken to study the relationships between previously identified mutations (u-shaped, serpent, hindsight and tailup) that selectively cause germband retraction defects in homozygous embryos, and a more pleiotropically acting locus, DER/faint little ball, the Drosophila Epidermal growth factor receptor. The former four loci are elements of at least two parallel and partially redundant cellular pathways that affect germband retraction by acting in amnioserosal development or maintenance. An additional discrete and unique pathway, represented by DER/faint little ball, is likely to function in the germband itself. While the role of the amnioserosa during germband retraction appears to be permissive, the action of DER in the germband may be mediated by the cytoskeleton (Goldman-Levi, 1996).

The amnioserosa is an extraembryonic, epithelial tissue that covers the dorsal side of the Drosophila embryo. The initial development of the amnioserosa is controlled by the dorsoventral patterning genes. A group of genes, referred to as the U-shaped-group (ush-group), is required for maintenance of the amnioserosa tissue once it has differentiated. Using several molecular markers, amnioserosa development was developed in the ush-group mutants: u-shaped (ush), hindsight (hnt), serpent (srp) and tail-up (tup). The amnioserosa in these mutants is specified correctly and begins to differentiate as in wild type. However, following germ-band extension, there is a premature loss of the amnioserosa. This cell loss is a consequence of programmed cell death (apoptosis) in ush, hnt and srp, but not in tup. Thus, the ush-group genes are implicated in the maintainance of the amnioserosa's viability. In light of these mutants' unretracted phenotype, the amnioserosa could be involved in signal reception or the initiation of signal transduction with respect to the adjacent ectoderm (Frank, 1996).

A large number of mutant alleles of ush have been isolated; these include hypomorphic mutants that affect the pattern of bristles on the head and thorax. Viable transallelic combinations of ush display additional dorsocentral and scutellar bristles on the notum and a loss of postverticle bristles on the head. Loss of function mutants affect the development of the dorsal half of the notum only, clones in the lateral part of the notum differentiate normally. Clones extending into the scutellum fail to differentiate, generating large gaps in this region. Clones touching the dorsal midline are associated with a cleft in the thorax, whereas clones extending into the dorsocentral area are associated with absence or abnormal positioning of the dorsocentral bristles. Remarkably, in almost all cases of mosaicism in the dorsocentral area, the dorsocentral bristles formed by wild-type cells are also found to be displaced from their normal postions. Therefore, there seems to be a nonautonomous effect of mutant cells at positions where wild-type bristles would be expected to form (Cubadda, 1997).

U-shaped and heart development

Multitype zinc-finger proteins of the class Friend of GATA/U-shaped (Ush) are known to function as transcriptional regulators of gene expression through their modulation of GATA factor activity. To better understand intrinsic properties of these proteins, the expression and function of the ush gene during Drosophila embryogenesis has been investigated. ush is dynamically expressed in the embryo, including several cell types present within the mesoderm. The gene is active in the cardiogenic mesoderm, and a loss of function results in an overproduction of both cardial and pericardial cells, indicating a requirement for the gene in the formation of these distinct cardiac cell types. Conversely, ectopic expression of ush results in a decrease in the number of cardioblasts in the heart and the inhibition of a cardial cell enhancer normally regulated by the synergistic activity of the Pannier and Tinman cardiogenic factors. These findings suggest that, similar to its known function in thoracic bristle patterning, Ush functions in the control of heart cell specification through its modulation of Pannier transcriptional activity. ush is also required for mesodermal cell migration early in embryogenesis, where it shows a genetic interaction with the Heartless fibroblast growth factor receptor gene. Taken together, these results demonstrate a critical role for the Ush transcriptional regulator in several diverse processes of mesoderm differentiation and heart formation (Fossett, 2000).

The ush gene exhibits a dynamic pattern of expression during embryogenesis. Gene transcripts are first detected at high levels in the primordium of the amnioserosa at stage 5. Additional expression is observed in germ band extending embryos, in cells of the developing anterior and posterior midgut, and in hemocyte precursors present in the cephalic mesoderm. By stage 11, ush RNA is detected in the dorsal ectoderm and in precursor cells of the hemocytes and fat body. By late embryogenesis, ush expression is greatly diminished, but transcripts are still observed in the dorsal ectoderm during dorsal closure and cells within, or associated with, the central nervous system. To investigate the possible expression of ush in mesodermal cells underlying the dorsal ectoderm, cross sections of embryos at stage 11 were examined. ush RNA is detected in a changing pattern in this germ layer, initially throughout most of the mesoderm and then in subpopulations of cells, including precursors of the fat body, visceral mesoderm, and cardiogenic mesoderm. Therefore, ush is expressed in the dorsal mesoderm, where it could function in the early stages of heart formation (Fossett, 2000).

pnr mutant embryos show a loss of contractile cardial cells and an overproduction of certain nonmuscle pericardial cells in the heart-forming region. To identify a possible role for ush in these cardiogenic processes, alterations in cardiac cell production were sought in mutant embryos. The ush alleles used in this analysis were ush¹ and ush², believed to be hypomorphic mutations of the gene, and Df(2L)al, a chromosome deletion that represents a ush null mutation. The D-mef2 heart enhancer-lacZ fusion gene serves as a cardial cell marker, since it is detected in progenitors of these cells around stage 11 and thereafter in two, then four cardioblasts per hemisegment of the forming dorsal vessel. Embryos homozygous for either a point mutation or deletion of the gene show an increase in the number of cells expressing the reporter gene, as compared with the wild-type embryo. In ush¹ embryos, a few hemisegments contain up to nine positive cells with an average of six cardial cells present in many clusters. In ush-deficiency embryos, a comparable increased density of cardial cells is found (Fossett, 2000).

Mef2 protein also marks cardioblasts; it is detected in the nuclei of all cardial, but not pericardial cells of the forming dorsal vessel. In wild-type embryos at stage 13, the germ band has retracted with cardioblasts migrating dorsally, separating from the dorsal somatic muscles. A lateral view at this stage shows a single row of cells that contains six stained nuclei per hemisegment. In contrast, ush mutant embryos possess supernumerary cardioblast nuclei. ush¹ and ush² embryos contain up to 12 nuclei per hemisegment with eight cells per cluster observed on average. Similar results have also been obtained with ush-deficiency embryos. Therefore, reducing or completely eliminating ush function leads to an increased production of cardial cells. Intriguingly, the ush heart phenotype uncovered by the analysis of these two markers directly contrasts the absence of cardial cells observed in pnr loss-of-function embryos (Fossett, 2000).

Production of pericardial cells was quantitated in ush mutant embryos, using Eve protein as a marker for a subset of these cells. In wild-type embryos at stage 12, there exist 11 Eve-positive clusters within the dorsal mesoderm, each containing about three cells. In contrast, the number of Eve-expressing pericardial cells increases in homozygous ush¹ and ush² embryos to an average of 5-6 per cluster. A similar increase in pericardial cell number is also observed in homozygous Df(2L)al embryos. Thus, ush gene activity is required to prevent the overproduction of this pericardial cell type, a function that has also been ascribed to the pnr gene (Fossett, 2000).

Because the loss of ush function resulted in a supernumerary cardial cell phenotype, the effect of expressing the gene throughout the mesoderm was monitored using the Gal4/UAS binary system. Mef2 was used to assess the status of cardial cells, with two contiguous rows of 52 cells present in the forming or mature dorsal vessel of wild-type embryos. In comparably staged embryos expressing ush throughout the mesoderm, a significant reduction in cardial cells is observed. The D-mef2 heart enhancer-lacZ fusion gene was used as a second marker for the cardial cells and also to assay the effect of ush expression on enhancer activity. In wild-type embryos at stage 16, the enhancer is active in eight cardial cells in most segments of the dorsal vessel. In contrast, beta-galactosidase activity is greatly diminished in the hearts of ush-expressing embryos, most likely a combination of the decrease in cardial cell number and the reduced activity of the D-mef2 cardiac enhancer. Thus, forced expression of ush has a potent negative effect on cardial cell formation and enhancer function (Fossett, 2000).

It has been shown that Pnr can function combinatorially with Tin in the regulation of the D-mef2 heart enhancer in Drosophila embryos. This synergistic activation was examined using a transient transfection assay in cultured Drosophila cells. The activation of a CAT reporter gene linked to the D-mef2 enhancer was monitored in cells transfected independently with Pnr, Tin, and Ush or with various combinations of the factors. The expression of Tin alone activated the enhancer about 2-fold above the basal level, whereas neither Pnr nor Ush affected enhancer activity. Coexpression of Pnr and Tin resulted in a synergistic activation of the enhancer to a level 5-6 times that of the basal activity, and this strong induction requires the binding of Tin to the Mef2 enhancer; a Tin DNA binding mutant, Tin (N-Q), failed to synergize with Pnr in the assay. In contrast, adding Ush as a third transfected factor significantly inhibits the synergistic activation of the enhancer by Pnr and Tin. This result demonstrates that Ush can antagonize the positive functional interaction of Pnr and Tin in the regulation of the cardial cell enhancer, which is consistent with the in vivo data (Fossett, 2000).

ush mutants contain an increased number of cardial cells. However, in about half of the embryos a disparity was noticed in the cardial cell populations, ranging from a high of 8-12 per hemisegment down to regions completely devoid of cells. This complex phenotype is observed with both ush hypomorphic and null alleles. This sporadic loss of cells from the dorsal-most part of the mesoderm is reminiscent of a htl mutant phenotype, where the absence of the encoded fibroblast growth factor receptor homolog results in an incomplete dorsal migration of mesodermal cells. In this event, cells fail to receive the ectodermal signal needed for their further commitment, resulting in a loss of dorsal mesodermal derivatives, including cardioblasts (Fossett, 2000).

To determine whether the variable absence of cardial cells in ush embryos is because of a cell migration defect, wild-type and mutant embryos were stained for Mef2 protein present in the invaginated population of mesodermal cells. In cross sections of normal embryos at stage 10, a uniform layer is observed where the dorsal-most mesodermal cells have migrated to a position adjacent to the dorsal-most ectodermal cells. In contrast, both htl and ush homozygous embryos display an irregular layer where mesodermal cells remain clustered and fail to undergo their complete dorsal migration. This result suggests ush function is required for the directional migration of the mesoderm. To investigate a potential genetic interaction of htl and ush in this process, embryos were examined that were heterozygous for mutations in each of the genes. These embryos also present a strong mesodermal migration phenotype, suggesting the two genes function in a common genetic pathway that controls this aspect of mesoderm differentiation. As was observed with homozygous ush embryos, slightly less than half of the transheterozygous embryos show a loss of cells from the dorsal mesoderm (Fossett, 2000).

It is thus thought that pnr has a dual requirement in the cardiogenic mesoderm because it is needed for the formation of cardial cells although simultaneously limiting the production of Eve-expressing pericardial cells. Based on these dissimilar phenotypes, it is postulated that Pnr might work with different combinations of factors to promote or repress the formation of cells within the distinct lineages. Recent studies have shown that Pnr and Tin act synergistically to induce cardial cells and activate gene expression, and the loss of function of either of these genes results in an absence of cardial cells. Therefore, the two work together in cardial cell specification (Fossett, 2000).

In contrast, Ush is a factor that normally suppresses cardial cell production. ush homozygous and hemizygous embryos show an increase in cardial cell number: the latter finding suggests Ush control of this cell population is dose-dependent, as is the case for Ush regulation of Pnr during sensory bristle development. Furthermore, forced expression of Ush decreases cardial cell production and D-mef2 heart enhancer activity, whereas ectopic expression of Pnr produces extra cardial cells and expands the domain of enhancer activity. Thus, Ush displays phenotypes that are in direct opposition to those of Pnr, suggesting that it can function as an antagonist of Pnr's cardiogenic activity. This conclusion is supported by the ability of Ush to inhibit the synergy of Pnr and Tin in the activation of the D-mef2 heart enhancer in cell transfection studies. As for the second cardiac phenotype, both pnr and ush are required to limit the number of Eve-expressing pericardial cells, consistent with a model in which Ush and Pnr function as corepressors in the control of these cells. To summarize, these genetic studies predict that, in the wild-type embryo, pnr is expressed and functions independent of ush in precursors of the cardioblast lineage. However, in neighboring cells that include the Eve lineage precursors, the expression and function of the two most likely overlaps. Future expression analyses of the two regulatory proteins at the resolution of single mesodermal cells will be required to elaborate on this genetic model in molecular terms (Fossett, 2000).

Friend of GATA (FOG) proteins regulate GATA factor-activated gene transcription. During vertebrate hematopoiesis, FOG and GATA proteins cooperate to promote erythrocyte and megakaryocyte differentiation. The Drosophila FOG homolog U-shaped (Ush) is expressed similarly in the blood cell anlage during embryogenesis. During hematopoiesis, the acute myeloid leukemia 1 homolog Lozenge and Glial cells missing are required for the production of crystal cells and plasmatocytes, respectively. However, additional factors have been predicted to control crystal cell proliferation. Ush is expressed in hemocyte precursors and plasmatocytes throughout embryogenesis and larval development, and the GATA factor Serpent is essential for Ush embryonic expression. Furthermore, loss of ush function results in an overproduction of crystal cells, whereas forced expression of Ush reduces this cell population. Murine FOG-1 and FOG-2 also can repress crystal cell production, but a mutant version of FOG-2 lacking a conserved motif that binds the corepressor C-terminal binding protein fails to affect the cell lineage. The GATA factor Pannier (Pnr) is required for eye and heart development in Drosophila. When Ush, FOG-1, FOG-2, or mutant FOG-2 is coexpressed with Pnr during these developmental processes, severe eye and heart phenotypes result, consistent with a conserved negative regulation of Pnr function. These results indicate that the fly and mouse FOG proteins function similarly in three distinct cellular contexts in Drosophila, but may use different mechanisms to regulate genetic events in blood vs. cardial or eye cell lineages (Fossett, 2001).

Using an antibody directed against Ush synthetic peptides, Ush protein was detected in an expression pattern similar to that of the gene transcript. Around embryonic stage 8, both ush RNA and protein can be detected in blood cell precursors. By stage 10, Ush-positive hemocyte precursors have spread throughout the lateral and ventral head mesoderm. As embryogenesis progresses, Ush is detected in stage 13 plasmatocytes migrating throughout the head mesoderm and down the ventral midline. During the late stages of embryogenesis, Ush continues to be expressed in plasmatocytes circulating throughout the embryonic hemolymph (Fossett, 2001).

lz expression in crystal cells is detected first during stage 10 and is maintained in this lineage until the late stages of embryogenesis. Fluorescent antibody staining and confocal microscopy were used to determine whether Ush and lz are coexpressed in the crystal cell lineage. To detect lz expression in hemocyte precursors and crystal cells, the expression of a UASlacZ reporter gene driven by lzGal4 (lzlacZ) was monitored. This reporter is active in hemocyte precursors as early as stage 10 and is expressed in the crystal cell lineage throughout embryogenesis. During embryonic stage 10, a number of hemocyte precursors express both Ush and lz. Later, during stage 13, the number of cells that expressed both lz and Ush decreases. Finally, during the late stages of embryogenesis, Ush is not detected in crystal cell lineage, evidenced by its failure to colocalize with the lzlacZ crystal cell marker. These results are consistent with a role for ush as a repressor of crystal cell production and suggest that ush expression is down-regulated in hemocyte precursors during crystal cell lineage commitment (Fossett, 2001).

During larval development, hematopoiesis takes place in the larval lymph glands, which flank the dorsal vessel. Plasmatocytes are specified and develop in the primary and secondary lobes of the gland, whereas crystal cells develop exclusively in the primary lobe. Ush is detected in most cells of primary and secondary lobes, consistent with expression in the plasmatocyte lineage. The protein is expressed in a differential pattern in the cells of the lymph glands, perhaps indicative of down-regulation during hemocyte precursor commitment. This may be analogous to the down-regulation of murine FOG-1 that is required for eosinophil and myeloid lineage differentiation (Fossett, 2001).

Srp function is required for hemocyte development and for differentiation of plasmatocytes and crystal cells. Furthermore, studies using amorphic alleles of srp indicate that it is required for hemocyte precursor specification. Srp is expressed first in the hemocyte precursors during embryonic stage 5, and, similar to Ush, its expression is maintained in plasmatocytes throughout embryogenesis. To determine whether an epistatic relationship exists between srp and ush, Ush expression was assayed in srp mutant embryos and Srp expression in ush mutant embryos. The hypomorphic allele srp³, which results in the production of hemocyte precursors, even with the reduction of Srp function, was used. In srp embryos, Ush is not detected in hemocyte precursors, plasmatocytes, or midgut, unlike the wild-type expression pattern. In contrast, Srp is observed in hemocyte precursors and plasmatocytes in both wild-type and ush mutant embryos. This result suggests ush resides downstream of srp in the hematopoiesis hierarchy and ush expression requires Srp function. Furthermore, ush is not required for the specification of hemocyte precursors or plasmatocytes, because these Srp-positive cells are detected in ush mutant embryos. Finally, wild-type levels of ush are present in the dorsal ectoderm of srp mutant embryos, indicating that dynamic ush expression is under the control of multiple regulators during embryogenesis (Fossett, 2001).

Previous studies have shown that ush functions to prevent the overproduction of sensory bristles, cardial cells, and pericardial cells. These observations, together with the findings that ush appears to be down-regulated during crystal cell lineage commitment, have suggested that Ush may act to limit crystal cell production. To test this hypothesis, assays were carried out for increased numbers of crystal cells in ush mutant embryos. Crystal cells are localized in a bilateral cluster of cells within the head mesoderm and require lz expression from embryonic stage 10 through 14 for their development. The lzlacZ genotype served as a crystal cell marker. Expression of lzlacZ was assayed in stage 13 embryos because during this stage the germ-band retraction phenotype can be used to distinguish ush mutants from wild-type embryos. Homozygous ush embryos show an increase in the number of lzlacZ-expressing cells compared with the wild-type control. These results were confirmed by using embryos harboring the Bc mutation, which renders crystal cells visible in late-stage homozygous embryos. Crystal cell production in ush Bc embryos was compared with the Bc parental strain, which has the wild-type ush allele. Again, homozygous ush Bc embryos have an increase in the number of crystal cells compared with the Bc embryos from the parental strain. Because the number of crystal cells in wild-type embryo populations can vary more than 2-fold, 20 wild-type and 20 ush embryos were sampled and a 30% overall increase in the number of crystal cells was seen by using either the lzlacZ or Bc marker. These results indicate that Ush functions to repress crystal cell production during hematopoiesis (Fossett, 2001).

To demonstrate further that Ush represses crystal cell production, Ush was expressed in crystal cells by using the Gal4/UAS binary system. The lzGal4 driver was used to express UASUsh in crystal cells, and their production was monitored by using the lzlacZ marker. Embryos with forced expression of Ush in crystal cells have a significant reduction in the number of these cells. Compared with similarly staged wild-type controls, UASUsh stage 13 and 16 embryos had a 30% and 85% reduction in number of crystal cells, respectively. A sample of 40 stage 13-16 UASUsh embryos averaged a 30% reduction in the number of crystal cells compared with wild-type controls. The phenotype of individual embryos within this population ranged from being completely devoid of crystal cells to wild-type cell numbers. These results indicate that down-regulation of ush during crystal cell lineage commitment is required for development of these cells. Together with the observed increase in crystal cell number in ush loss-of-function assays, these findings suggest that Ush functions during hematopoiesis to limit the number of hemocyte precursors that enter the crystal cell lineage (Fossett, 2001).

Recent studies have indicated that FOG proteins may function to regulate the commitment of several hematopoietic lineages. Ectopic expression of FOG proteins mFOG-1, mFOG-2, and xFOG (Xenopus FOG) early in Xenopus development represses red blood cell formation, possibly by down-regulating Gata-1 expression. These data suggest that FOG proteins may act to limit the differentiation of erythrocytes to prevent depletion of pluripotent stem cells. Furthermore, by using an in vitro avian hematopoietic differentiation system, it has been shown that FOG-1 represses eosinophil-specific gene expression and that forced expression of FOG-1 in eosinophils produces a multipotent precursor phenotype. Thus, down-regulation of FOG-1 in multipotent hematopoietic precursors is an essential step in eosinophil differentiation. In addition to findings that misexpressed Ush repressed crystal cell production, FOG-1 and FOG-2 also repress crystal cell number, indicating that the mechanism by which these proteins limit crystal cell number may be conserved. Taken together, studies using the Drosophila and vertebrate systems suggest that FOG proteins function to preserve the multipotent hemocyte precursor pool by controlling the lineage commitment of specific cell types (Fossett, 2001).

An additional factor that may be required to control lineage commitment is CtBP. This transcriptional corepressor may interact with FOG-1 and FOG-2 to repress erythrocyte differentiation, because a mutant version of FOG-2 lacking the consensus PXDL sequence fails to repress erythrocyte differentiation when ectopically expressed during Xenopus development. CtBP may be required for FOG protein repression of crystal cell production. It is noteworthy that CtBP likely functions during Drosophila hematopoiesis because a lacZ reporter gene inserted in the enhancer region of the CtBP gene is expressed in the larval plasmatocyte lineage. Thus, the FOG and CtBP class of transcriptional regulators may act together to control hemocyte lineage commitment in a pathway that is conserved evolutionarily (Fossett, 2001).

FOG function involves binding to its GATA partner's N-terminal zinc finger. Srp is the only known hematopoietic GATA factor in Drosophila and reportedly contains a single C-terminal zinc finger. However, a survey of the srp genomic sequence shows an ORF within the third intron of the gene that putatively encodes an N-terminal zinc finger with 96% homology to that of Pnr. This raises the possibility that Ush interacts with an alternatively spliced isoform of Srp during hematopoiesis (Fossett, 2001).

Ush appears to negatively regulate the cardiogenic function of the GATA-4 homolog Pnr, converting Pnr from a transcriptional activator to a repressor as observed during sensory bristle development. As with Ush, forced mesodermal expression of FOG-1, FOG-2, and DeltaFOG-2 (lacking a conserved motif that binds the corepressor C-terminal binding protein) also produces a diminution of cardial cells. These results demonstrate a functional conservation of the FOG proteins during Drosophila cardiogenesis, which most likely involves negative regulation of the cardiogenic activity of Pnr. In addition, forced expression of FOG proteins disrupts eye development-producing phenotypes that mimic pnr loss of function mutants, presumably by repressing Pnr activation of its downstream effector genes (Fossett, 2001).

The disruption of eye development and the repression of cardial cell production by FOG proteins occurred in the absence of the CtBP-binding motif. This is consistent with the work that shows that FOG-1 and FOG-2 repression of GATA-4 activation of cardiac promoters does not require the corepressor CtBP. Rather, conserved N-terminal regions of the murine FOG proteins were required for the repression of GATA-4 transcriptional activation, indicating that an alternative repressor mechanism may be used to negatively regulate GATA-4. An emerging hypothesis suggests that CtBP may be a hematopoietic corepressor, and an alternative corepressor may be required during heart development. Results showing that DeltaFOG-2 does not repress crystal cell production but does repress cardial cell production is evidence for this dual mechanism of FOG gene regulation during heart development and hematopoiesis in a single experimental organism (Fossett, 2001).

In conclusion, Ush and Lz function antagonistically during crystal cell lineage commitment and Ush is required to limit the overproliferation of crystal cells. This demonstrates a possible intersection between the FOG and AML-1 gene pathways, which may prove important for understanding vertebrate hematopoiesis. Furthermore, this study expands the molecular characterization of the earliest events of hematopoiesis in Drosophila, identifying additional conserved genes that establish the fly as a model organism for hematopoiesis (Fossett, 2001).

Inductive signaling is of pivotal importance for developmental patterns to form. In Drosophila, the transfer of TGFß (Dpp) and Wnt (Wg) signaling information from the ectoderm to the underlying mesoderm induces cardiac-specific differentiation in the presence of Tinman, a mesoderm-specific homeobox transcription factor. Evidence that the Gata transcription factor, Pannier, and its binding partner U-shaped, also a zinc-finger protein, cooperate in the process of heart development. Loss-of-function and germ layer-specific rescue experiments suggest that pannier provides an essential function in the mesoderm for initiation of cardiac-specific expression of tinman and for specification of the heart primordium. u-shaped also promotes heart development, but unlike pannier, only by maintaining tinman expression in the cardiogenic region. By contrast, pan-mesodermal overexpression of pannier ectopically expands tinman expression, whereas overexpression of u-shaped inhibits cardiogenesis. Both factors are also required for maintaining dpp expression after germ band retraction in the dorsal ectoderm. Thus, it is proposed that Pannier mediates as well as maintains the cardiogenic Dpp signal. In support, it is found that manipulation of pannier activity in either germ layer affects cardiac specification, suggesting that its function is required in both the mesoderm and the ectoderm (Klinedinst, 2003).

pnr and ush are both expressed in the mesoderm at the time of cardiac mesoderm formation, in addition to their expression in the dorsal ectoderm. Mesodermal expression of pnr is restricted to the dorsal cardiogenic margin, whereas ush extends more laterally. In order to assess the requirement for pnr and ush in initiating cardiac mesoderm and cardiac cell type-specific differentiation, tin expression was examined at progressively later developmental stages in null mutants for both pnr and ush. During mid-stage 11, tin is expressed segmentally in two regions of the mesoderm. The dorsal clusters of cells correspond to the cardiac precursor cells, whereas the lateral clusters will become part of the visceral mesoderm. In same stage pnr mutant embryos, tin expression is dramatically reduced in the clusters that correspond to the cardiac precursors, indicating that cardiogenesis is not being initiated. tin expression in the visceral mesodermal clusters, as well as tin expression earlier in development, is unaffected, suggesting the heart is a focal point for pnr function, which is consistent with its cardiac-restricted expression in the mesoderm. By contrast, ush mutant embryos initially seem to exhibit normal tin expression. At later stages, when tin expression is solely restricted to the heart cells, ush mutants display a progressively more severe reduction in tin expression, approaching the phenotype of pnr mutants. Thus, both pnr and ush are required for heart-specific tin expression, although ush seems to be initially dispensable (Klinedinst, 2003).

Even though tin is initially expressed in all heart progenitors, its expression is later turned off in some specific lineages, but continues to be expressed in many myocardial and pericardial cells. To determine which heart cells are affected in pnr and ush mutants, mutant embryos were examined with various markers. eve, for example, is co-expressed with tin in 11 clusters of heart progenitors, and these lineages give rise to a subset of pericardial cells. eve expression is only moderately reduced in pnr and hardly at all in ush mutants at early as well as later stages; this is accompanied by patterning defects at progressively later stages. By contrast, the lbe-expressing heart progenitors, which produce both myocardial and pericardial cells, are dramatically reduced in pnr but less so in ush mutants. Moreover, the svp-expressing cells, which also give rise to a mixed lineage, but cease to co-express tin at later stages, are dramatically reduced in both mutants. Thus, all lineage markers assayed are reduced in both mutants, but each is affected with disproportional severity, which is consistent with the idea that the formation of each cell type has a direct requirement for pnr and ush (Klinedinst, 2003).

Both tin and pnr have been shown to be targets of Dpp signaling at stage 9/10. It is proposed that dpp is necessary again at stage 11 to activate and maintain pnr and tin expression in the cardiogenic region of the mesoderm. First, pnr is activated with the help of early stage 11 tin, which is expressed broadly throughout the dorsal mesoderm, and dpp, which is expressed in a narrow dorsal ectodermal stripe. Then, at mid-stage 11, tin is restricted to the cardiogenic region with the help of mesodermal pnr as well as continuous ectodermal Dpp signaling. Once both are activated in the cardiogenic mesoderm, they are likely to contribute to the maintenance of each other's expression, probably aided again, but only moderately, by ectodermal Dpp signaling. This interpretation is consistent with mesodermal versus ectodermal expression of dominant-negative pnrEnR and the dpp target repressor encoded by brk. They are both equally effective in reducing cardiac-specific tin when expressed in the mesoderm, but ectodermal repression is more effective when dorsal-stripe dpp at stage 11 is also affected (as in the case of ZKr-Gal4>UAS-brk, but not with ZKr-Gal4>UAS-pnrEnR) (Klinedinst, 2003).

Mesodermal overexpression of ush and co-overexpression with pnr results in a decrease in the amount of cardiac-specific tin expression, suggesting that ush may not only be required along with pnr for heart development, but also play an inhibitory role. To test this hypothesis further, pnrD4, an allele that abolishes Ush binding to Pnr was overexpressed; not only ectopic tin expression was found at early stages of cardiogenesis, but also undiminished and even increased levels of expression at later stages. A similar phenotype was observed when both pnrD4 and ush were expressed throughout the mesoderm, suggesting that ush plays an anti-cardiogenic role by antagonizing the activity of wild-type Pnr, but not that of PnrD4. It would be interesting to see if pan-mesodermal overexpression of wild-type pnr in a ush mutant background results in ectopic tin expression similar to pnrD4, or if a minimal amount of ush activity is required to maintain normal and ectopic tin expression even with forced pnr expression. Interestingly, overexpression of both pnr and tin together in the mesoderm also causes a pnrD4-like phenotype, as assayed with Hand expression, suggesting that pnr and tin collaborate during initiation and subsequent differentiation of the heart progenitors (Klinedinst, 2003).

Although in vitro the Ush-related FOG factors are primarily known for their role as transcriptional repressors, they apparently can also function as co-activators: Fog2 can synergistically activate or repress the transcriptional activity of Gata4, depending on the (cardiac) promoter and cell line used, and FOG-1 can cooperate with Gata1 to transactivate NF-E2, an erythroid cell-specific promoter. Moreover, the ventricular hypoplasia and other heart defects observed in Fog2-deficient mice suggest a deficit rather than an excess in heart development. In addition, mice with an equivalent mutation to PnrD4 knocked into the Gata4 locus, thus eliminating binding to Fog2, exhibit in many ways a similar phenotype to Fog2-deficient mice. These data are consistent with the idea that Fog2 is normally involved in promoting rather than antagonizing cardiogenesis, similar to what was found with genetic studies during Drosophila heart development (Klinedinst, 2003).

The dual role of Ush suggests that the amount of Ush may be crucial for whether it exerts its function as a an activator or repressor, perhaps by binding to different sets of co-factors in a concentration-dependent manner. Alternatively, the mode of transcriptional regulation by Ush could be stage-dependent: at stage 11, Pnr and Ush cooperate as transcriptional activators in initiating cardiac-specific tin expression and heart development, but later Ush becomes a repressor to limit the transcriptional activation of tin by Pnr (Klinedinst, 2003).

pnr and ush are initially broadly expressed in the dorsal ectoderm of the early embryo, but by germband retraction the ectodermal expression of pnr is confined to a narrow stripe of cells along the border of the amnioserosa, which overlaps with the thin dorsal dpp stripe. The early ectodermal expression of ush is restricted to the presumptive amnioserosa, and by germband extension, ush also overlaps with the dorsalmost region of the ectoderm. These patterns of expression suggest that pnr and ush may be acting in both germ layers. The genetic data, including germ layer-specific expression of wild-type and dominant-negative pnr constructs, as well as germ layer-specific rescue experiments suggest strongly that pnr and ush function is not only needed in the mesoderm, but also in the ectoderm for heart formation. The ectodermal requirement for pnr and ush in heart development is probably achieved via the maintenance of dpp expression, since dorsal stripe dpp expression diminishes in pnr and ush mutants and ectodermal interference with pnr, ush and/or dpp-signaling function compromises the normal progression of heart development (Klinedinst, 2003).

U-shaped and leading edge cells

The leading edge (LE) is a single row of cells in the Drosophila embryonic epidermis that marks the boundary between two fields of cells: the amnioserosa and the dorsal ectoderm. LE cells play a crucial role in the morphogenetic process of dorsal closure and eventually form the dorsal midline of the embryo. Mutations that block LE differentiation result in a failure of dorsal closure and embryonic lethality. How LE cells are specified remains unclear. To explore whether LE cells are specified in response to early dorsoventral patterning information or whether they arise secondarily, the extent of amnioserosa and dorsal ectoderm was altered genetically, and LE cell fate was assayed. No expansion of LE fate is observed in dorsalized or ventralized mutants. Furthermore, the LE fate arises as a single row of cells, wherever amnioserosa tissue and dorsal epidermis are physically juxtaposed. Taken together these data indicate that LE formation is a secondary consequence of early zygotic dorsal patterning signals. In particular, proper LE specification requires the function of genes such as u-shaped and hindsight, which are direct transcriptional targets of the early Decapentaplegic/Screw patterning gradient, to establish a competency zone from which LE arises. It is proposed that subsequent inductive signaling between amnioserosa and dorsal ectoderm restricts the formation of LE to a single row of cells (Stronach, 2001).

Using mutations that influence DV patterning, it is possible to alter the size and distribution of BMP target gene expression patterns, which indicate the extent of amnioserosa and dorsal ectodermal cell fates. If LE fate was specified directly by a particular threshold level of BMP signal, then one would expect LE fate to be perturbed in concert with amnioserosa and dorsal ectoderm fates in DV mutants. Mutations in genes such as dorsal, Toll, brinker and short gastrulation alter the size of BMP target gene expression domains; however, these mutants failed to alter specification of LE fate. Among these genotypes, brk and sog specifically modulate the shape of the BMP signaling gradient in a region where LE fate might arise, yet LE formation in these mutants is fundamentally normal. Furthermore, in dorsalized embryos, LE cells were observed regularly at the boundary between amnioserosa and dorsal ectoderm even when the morphology of these tissues was severely disrupted. Islands of amnioserosa cells within a field of ectoderm were consistently surrounded with a single row of LE cells, independent of the number of amnioserosa cells constituting the island. The converse situation also occurred; again, a single row of LE cells formed at the boundary between the ectoderm and amnioserosa (Stronach, 2001).

DV mutants were also analyzed to determine whether a decrease in BMP signaling activity converts amnioserosa to LE as predicted by a gradient patterning model. A range of ventralizing mutations (cactus, sog, screw, dpp) displaying progressive loss of amnioserosa tissue did not give rise to embryos with an expanded domain of LE cells. In fact, LE cells were not detected in the absence of amnioserosa. No situation was found in which an altered BMP gradient was associated with expanded LE fate, thus the prediction of a direct gradient response model does not explain LE fate specification (Stronach, 2001).

Notably, DV mutant embryos that perturb the BMP gradient, also perturb the expression domains of target genes, including u-shaped and hindsight, without altering LE specification. However, loss of ush and hnt function results in specific and distinct perturbations in LE formation. Thus, the interpretation that LE fate specification is not a direct early response to the BMP gradient is favored, but rather is a secondary consequence of the specification of dorsal fates through the action of BMP target genes like ush and hnt (Stronach, 2001).

Taken together, these results raise the possibility that amnioserosa may be required for LE formation. To address the function of amnioserosa for LE specification, puc enhancer expression was examined in several mutants of the U-shaped class, including u-shaped (ush) and hindsight (hnt). Incidentally, the dorsal expression domains of these genes are directly regulated by DV patterning signals. In these mutant embryos, the amnioserosa tissue is fated normally and begins to differentiate up to stage 11, but then degenerates prematurely. In both ush and hnt mutants, programmed cell death takes place over the course of a few hours, with elimination of amnioserosa cells by stage 13 -- the time when dorsal closure would normally commence. Unexpectedly, different patterns of expression were observed with the puc enhancer in the two mutants. In ush embryos, ß-gal-positive cells were not detected. In contrast, hnt mutant embryos displayed Puc-positive LE cells at the edge of the dorsal ectoderm, albeit with less uniform expression than normally observed. To confirm these observations, the accumulation of dpp mRNA in the LE was examined. Similar to puc enhancer expression, differential expression of dpp was observed in ush versus hnt mutant embryos. ush mutant embryos show a consistent and significant reduction in LE dpp expression, although residual dpp transcripts are seen. dpp expression appears relatively normal in hnt mutant embryos (Stronach, 2001).

In addition to the differential expression of two LE markers in the U-shaped mutants, ectopic expression of LE markers is observed only in hnt mutant embryos. ß-gal-positive cells were observed in the region of the amnioserosa in hnt mutants as early as stage 11, raising the possibility that this could be an example of expanded LE cell fates. These cells adopt only partial LE cell fate, for the following reasons. These cells do not express the LE marker Fasciclin III, but do express two other LE molecules, albeit aberrantly. puc, for example, is expressed precociously in these cells, preceding Fasciclin III expression in the ectoderm, and dpp is rarely but reproducibly expressed. Additionally, cells in this region express amnioserosa fate markers such as race, through stage 11. Thus, based on the possibility that these cells may co-express LE and amnioserosa markers during stage 11, their identity cannot be unequivocally determined. These results may indicate that these cells are of mixed fate. The presence of ectopic LE-like cells in hnt mutant embryos, coupled with the severe reduction of LE fate markers in ush mutants, suggest that the distinction between amnioserosa and LE is a secondary consequence of Hnt and Ush functions, not a direct result of specific BMP signaling thresholds (Stronach, 2001).

If LE cells are specified as a secondary consequence of DV patterning gradients, then what additional mechanisms are at work to define LE as a single row of cells? The data are consistent with several mechanisms. One possibility is that specification of the LE involves the combinatorial action of nested sets of transcriptional regulators, including Hnt dorsally and Ush in a broader domain. Accordingly, loss of Hnt function is predicted to result in a failure to differentiate amnioserosa, coupled with dorsal expansion of more lateral fates, such as the LE. Consistent with this model, hnt mutant embryos display Puc-positive cells with partial LE character in the region of the dying amnioserosa during stage 11. These results suggest that Hnt may be necessary to distinguish amnioserosa from LE fate at the time of extended germ band stage. This timing is late, relative to the timing of the early BMP threshold response, further supporting the notion that LE specification is a secondary consequence of initial BMP signaling (Stronach, 2001).

Ush may play a role in differentiation of more lateral fates adjacent to the amnioserosa and the Hnt expression domain. Indeed, Ush function is essential for LE development because LE does not form in ush mutant embryos. Based on these results, it is imagined that Ush could define a competency zone from which LE cells arise, or Ush could participate in generating or modulating a signal(s) for communication between the differentiating amnioserosa and dorsal ectoderm. Ush is related to mammalian zinc-finger protein family, Friend of GATA (FOG), which has been shown to participate as a cofactor with GATA transcription factors. Together, these protein complexes regulate cell fate determination multiple times during both mammalian and Drosophila development. Interestingly, FOG2, a mammalian homolog of Ush, appears to be required during an inductive signaling event between two distinct tissues in the mouse heart, suggesting that inductive processes in development may commonly use the function of Ush family members. It has not been determined whether the function of Ush in LE cell specification is localized to the amnioserosa, the dorsal ectoderm, or both. Experiments to replace Ush function in a tissue-specific manner should address this issue (Stronach, 2001).

Although transcriptional targets of BMP signaling, such as ush and hnt, among others, define at least three specific threshold responses, the size difference between the nested expression domains of these markers still fails to account for a cell fate defined by a single row of cells. An additional mechanism to explain the spatially restricted stripe of LE cells is through an inductive signaling event. From the analysis of dorsalized mutants, it is observed that LE forms as a result of the juxtaposition of amnioserosa tissue with dorsal ectoderm, which may provide spatially limited activation of the JNK pathway. Thus, restricted expression of JNK target genes, such as puc and dpp may be a direct result of a signal that specifies LE (Stronach, 2001).

Communication between the amnioserosa and the dorsal ectoderm during embryogenesis has been suggested in two cases recently: (1) Hnt expression in the amnioserosa is required nonautonomously for proper cell rearrangements in the dorsal ectoderm, associated with retraction of the embryonic germband; (2) the raw gene product appears to be expressed in the amnioserosa, though it influences the activity of the JNK pathway in the ectoderm during dorsal closure. As amnioserosa and ectoderm develop, they may acquire different cell affinities, which cause them to sort into separate domains or islands (in the case of dorsalized embryos), displaying smooth borders at their interface. A difference in cell adhesion at the boundary may be sufficient to generate signaling for LE specification similar to inductive mechanisms at work at the compartmental boundaries of larval imaginal discs. The challenge now will be to identify molecules that may participate in an inductive signal (Stronach, 2001).

These results suggest that a multistep process determines the LE as a single row of cells. LE does not form directly in response to discrete intermediate levels of BMP signaling activity, but forms secondarily by the action of transcriptional regulators that are themselves BMP target genes. Among these targets, Hnt and Ush define a LE competency zone that is expanded in hnt mutants and eliminated in ush mutants. It is proposed that from within the competency zone, LE fate is further refined to a single row by an unknown inductive signal generated by the physical juxtaposition of amnioserosa with dorsal ectoderm. This signal activates the JNK pathway that regulates localized expression of dpp and puc (Stronach, 2001).

U-shaped protein domains required for repression of cardiac gene expression in Drosophila

U-shaped is a zinc finger protein that functions predominantly as a negative transcriptional regulator of cell fate determination during Drosophila development. In the early stages of dorsal vessel formation, the protein acts to control cardioblast specification, working as a negative attenuator of the cardiogenic GATA factor Pannier. Pannier and the homeodomain protein Tinman normally work together to specify heart cells and activate cardioblast gene expression. One target of this positive regulation is a heart enhancer of the Drosophila mef2 gene and U-shaped has been shown to antagonize enhancer activation by Pannier and Tinman. Protein domains of U-shaped required for its repression of cardioblast gene expression were mapped. Such studies showed GATA factor interacting zinc fingers of U-shaped are required for enhancer repression, as well as three small motifs that are likely needed for co-factor binding and/or protein modification. These analyses have also allowed for the definition of a 253 amino acid interval of U-shaped that is essential for its nuclear localization. Together, these findings provide molecular insights into the function of U-shaped as a negative regulator of heart development in Drosophila (Tokusumi, 2007).

Through the use of an established assay to monitor Pannier-dependent cardioblast gene activity, and the generation and analysis of 20 different versions of the U-shaped protein, six U-shaped domains required for its repression of mef2 gene expression were identified. Three previously identified GATA-interacting zinc fingers of U-shaped are critical for this inhibitory property, which likely reflects the necessity of multiple zinc fingers forming a strong and stable interaction with the Pannier GATA factor. Whether Pannier-U-shaped complex formation interferes with the physical interaction of Pannier and Tinman in the synergistic activation of D-mef2 target sequences remains to be determined (Tokusumi, 2007).

U-shaped may also directly antagonize Pannier function as has been shown in the process of sensory bristle formation. Heterodimerization of U-shaped with Pannier converts the GATA transcriptional activator into a transcriptional repressor, an event that leads to the non-activation of target genes such as ac, sc, and wg in the dorsal notum of the wing disc. It is noteworthy that the results demonstrated the requirement of a binding site for the CtBP transcriptional co-repressor protein. In the context of the cardiogenic mesoderm, the combination of Pannier, U-shaped, and CtBP may prevent mesodermal cells from initiating gene expression programs needed for the specification of the cardioblast fate. In contrast, the combination of Pannier, Dorsocross, and Tinman is known to activate a regulatory network programming heart cell specification and cardioblast differentiation. Additional studies will be needed to elucidate the potential role of CtBP as an antagonist of cardiac gene expression and heart development. If U-shaped-CtBP interaction plays a crucial inhibitory role, then one would predict comparable dorsal vessel phenotypes for CtBP and U-shaped in loss- and gain-of-function genetic backgrounds (Tokusumi, 2007).

Finally, these studies have defined a 253 amino acid region required for nuclear localization of U-shaped. Within this interval, two highly basic amino acid sequences have been defined as being essential for U-shaped ability to inhibit Pannier-mediated cardiac gene expression. Perhaps, these motifs are required to facilitate the binding and stable interaction of co-repressor proteins with U-shaped. Another possibility is that these sequences serve as sites for post-translational modification, such as acetylation and/or methylation. Selective protein modification(s) may be a requisite for U-shaped to act as a negative modulator of Pannier transcription factor function during cardiogenesis in Drosophila (Tokusumi, 2007).

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date revised: 15 January 2008 

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