tinman

Promoter

Genetic analyses indicate that tinman resides downstream of the mesodermal determinant twist, which encodes a basic helix-loop-helix-type transcription factor. However, the regulation of tinman by twist remains poorly understood. Using expression assays in cultured cells and transgenic flies, it has been shown that two distinct clusters of E-box regulatory sequences, present upstream of the tinman gene, mediate tinman expression in the visceral mesoderm. These elements are conserved between the Drosophila melanogaster and Drosophila virilis tinman genes and serve as binding sites for Twist (E1 cluster located from -1134 to -1101) and Tinman (E2 cluster located from -868 to -831) proteins. In cultured cells, Twist and Tinman binding results in the activation of tinman gene expression. In transgenic animals, the E1 and E2 clusters are functionally connected; both elements are required for tinman activation in cells of the visceral mesoderm and also for tinman repression in cells of the somatic musculature. These results demonstrate that tinman is a direct transcriptional target for Twist and its own gene product in visceral mesodermal cells, supporting the idea that twist and tinman function in the subdivision of the mesoderm during Drosophila embryogenesis (Lee, 1997).

The Drosophila tinman homeobox gene has a major role in early mesoderm patterning: it determines the formation of visceral mesoderm, heart progenitors, specific somatic muscle precursors and glia-like mesodermal cells. These functions of tinman are reflected in its dynamic pattern of expression, which is characterized by initial widespread expression in the trunk mesoderm, then refinement to a broad dorsal mesodermal domain, and finally restricted expression in heart progenitors. Each of these phases of expression is driven by a discrete enhancer element, the first being active in the early mesoderm, the second in the dorsal mesoderm and the third in cardioblasts. Surprisingly, all of these elements are located at positions downstream of the transcription start site. Element B(1800 bp) is located in the first intron; a second enhancer element, D (about 350bp), is located about 2 kb downstream of the 3' end of tin and activates gene expression in the dorsal portion of the mesoderm. Element D is active between stage 11 and early stage 12 of embryogenesis. A third element, C (300bp) is active in the dorsal vessel. This element activates expression from stage 12 on, in four out of six cardioblasts per hemisegment. Finally, an element A (about 500 bp), located in the 5' portion of the first intron, activates tin in the anterior tip of the head. After invagination of the stomodeum, the bulk of these tin expressing cells form the roof of the pharynx (Yin, 1997).

The early-active enhancer element is a direct target of twist, a gene necessary for tinman activation that encodes a basic helix-loop-helix (bHLH) protein. This 180 bp enhancer includes three E-box sequences that bind Twist protein in vitro and are essential for enhancer activity in vivo. Ectodermal misexpression of twist causes ectopic activation of this enhancer in ectodermal cells, indicating that twist is the only mesoderm-specific activator of early tinman expression. The 180 bp enhancer also includes negatively acting sequences. Binding of Even-skipped to these sequences appears to reduce twist-dependent activation in a periodic fashion, thus producing a striped tinman pattern in the early mesoderm. In addition, these sequences prevent activation of tinman by twist in a defined portion of the head mesoderm that gives rise to hemocytes. This repression requires the function of buttonhead, a head-patterning gene: buttonhead is necessary for normal activation of the hematopoietic differentiation gene serpent in the same area. The second expression domain, restricting tin mRNA expression in the dorsal mesoderm, is triggered by Dpp-mediated induction events. Together, these results show that tinman is controlled by an array of discrete enhancer elements that are activated successively by differential genetic inputs, as well as by closely linked activator and repressor binding sites within an early-acting enhancer, which restricts twist activity to specific areas within the twist expression domain (Yin, 1997).

The embryonic dorsal vessel in Drosophila possesses anteroposterior polarity and is subdivided into two chamber-like portions, the aorta in the anterior and the heart in the posterior. The heart portion features a wider bore as compared with the aorta and develops inflow valves (ostia) that allow the pumping of hemolymph from posterior toward the anterior. Homeotic selector genes provide positional information that determines the anteroposterior subdivision of the dorsal vessel. Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) are expressed in distinct domains along the anteroposterior axis within the dorsal vessel, and, in particular, the domain of abd-A expression in cardioblasts and pericardial cells coincides with the heart portion. Evidence is provided that loss of abd-A function causes a transformation of the heart into aorta, whereas ectopic expression of abd-A in more anterior cardioblasts causes the aorta to assume heart-like features. These observations suggest that the spatially restricted expression and activity of abd-A determine heart identities in cells of the posterior portion of the dorsal vessel. Abd-B, which at earlier stages is expressed posteriorly to the cardiogenic mesoderm, represses cardiogenesis. In light of the developmental and morphological similarities between the Drosophila dorsal vessel and the primitive heart tube in early vertebrate embryos, these data suggest that Hox genes may also provide important anteroposterior cues during chamber specification in the developing vertebrate heart (Lo, 2002).

Antp is strongly expressed in four consecutive pairs of cardioblasts in the anterior of the dorsal vessel. The three anterior cardioblast pairs of this domain of strong Antp expression are the posterior three tinman (tin) cardioblast pairs of segment A1, while the fourth pair corresponds to the anterior pair of the two seven up (svp) cardioblast pairs located between A1 and A2. There is also strong expression in at least six pericardial cells flanking the domain of strong cardioblast expression, all of which are non-Tin expressing pericardial cells. Weaker Antp expression is seen in a row of three or four consecutive cardioblast pairs in T3 immediately anterior to the domain of strong Antp, and also in the four tin cardioblast pairs of segment A2 (Lo, 2002).

Since abd-A expression coincides with the heart portion of the dorsal vessel, tests were made to see whether it acts to specify the cardioblasts in which it is expressed to eventually form the heart. In order to distinguish aorta cardioblasts from heart cardioblasts, two different molecular markers were utilized. The first marker was the pattern of ß-Gal derived from the tinCdelta5-lacZ transgene, where the expression of a lacZ gene is controlled by an internally deleted tinman cardiac enhancer element, tinCdelta5. This element drives ß-Gal expression in all the cardioblasts of the aorta, whereas in the heart it is only expressed in three segmentally-spaced double pairs of cardioblasts. These particular cardioblasts correspond to the svp cardioblasts of the heart. The second marker is wingless (wg), which is expressed in these same three double pairs of svp cardioblasts within the heart of the late embryonic dorsal vessel (Lo, 2002).

In abd-A null mutant embryos, the pattern of tinCdelta5-lacZ-derived ß-Gal is continuous in the heart as well as in the aorta of the dorsal vessel. In addition, it appears that the width of the heart is now the same as that of the aorta when compared with a wildtype embryonic dorsal vessel. Similarly, the late expression of Wg in the svp cardioblasts of the heart is not detectable in these mutant embryos. The alterations in the pattern of these two markers strongly suggest that heart cardioblasts have not been specified in the posterior of the dorsal vessel of abd-A null mutant embryos and that these posterior cardioblasts have been transformed instead into aorta cardioblasts. This would indicate that abd-A is necessary for the specification of heart cardioblasts in the posterior portion of the dorsal vessel where it is normally expressed (Lo, 2002).

Transcriptional Regulation

tinman expression, except for a patch in the head, is dependent on twist function. In contrast, snail, another mesoderm determinant does not appear to be required for tinman initiation, but is necessary for the maintenance of tinman expression after germband elongation (Bodmer, 1990).

After gastrulation, progenitor cells of the cardiac, visceral and body wall musculature arise at defined positions within the mesodermal layer of the Drosophila embryo. An early and important event in the regional subdivision of the mesoderm is the restriction of tinman expression to dorsal mesodermal cells. Genetic analysis has shown that this homeobox gene controls the formation of the visceral musculature and the heart from dorsal portions of the mesoderm. An inductive signal from dorsal ectodermal cells is required for activation of tinman in the underlying mesoderm. Decapentaplegic serves as a signaling molecule in this process. The spatial expression of dpp in the ectoderm determines which cells of the mesoderm become competent to develop into visceral mesoderm and the heart (Frasch 1995).

The homeobox gene tinman plays a key role in the specification of Drosophila heart progenitors and the visceral mesoderm of the midgut, both of which arise at defined positions within dorsal areas of the mesoderm. In addition to the heart and midgut visceral mesoderm, tinman is also required for the specification of all dorsal body wall muscles. Thus it appears that the precursors of the heart, visceral musculature, and dorsal somatic muscles are all specified within the same broad domain of dorsal mesodermal tinman expression. Locally restricted activities of tinman are also observed during its early, general mesodermal expression, where tinman is required for the activation of the homeobox gene buttonless in precursors of the dorsal median glial cells along the ventral midline. These observations, together with others showing only mild effects of ectopic tinman expression on heart development, indicate that tinman function is obligatory, but not sufficient to determine individual tissues within the mesoderm. Therefore, it is proposed that tinman has a role in integrating the positional information that is provided by intersecting domains of additional regulators and signals, which may include Wingless, Sloppy Paired, and Hedgehog in the dorsal mesoderm and EGF-signaling at the ventral midline. Previous studies have shown that Dpp acts as an inductive signal from dorsal ectodermal cells to induce tinman expression in the dorsal mesoderm, which, in turn, is needed for heart and visceral mesoderm formation. In the present report, Thickveins, a type I receptor of Dpp, is shown to be essential for the transmission of Dpp signals into the mesoderm. Constitutive activity of Tkv in the entire mesoderm induces ectopic tinman expression in the ventral mesoderm, and this results in the ectopic formation of heart precursors in a defined area of the ventrolateral mesoderm (Yin, 1998).

Because of the crucial role of dpp in inducing dorsal mesodermal tinman expression and the specification of dorsal mesodermal tissues, it is of interest to determine whether other components known to function in dpp-mediated signaling events during blastoderm are also required for mesoderm induction. Screw, a second BMP2/4-related gene product, Tolloid, a BMP1-related protein, and the zinc finger-containing protein Schnurri, are all shown to be required to allow full levels of tinman induction during this process. screw, which encodes a secod BMP2/4-related molecule, has been proposed to act synergistically with dpp to specify dorsal ectoderm and amnioserosa. Similarly, it has been shown that tolloid, which encodes a BMP1-related metalloproteinase, acts to enhance the activity of the dpp gene product during mesoderm induction. Both scw and tolloid are shown to be required for normal induction of tinman expression in the dorsal mesoderm, and in the absence of either gene activity, tinman expression in the dorsal mesoderm is reduced and segmentally interrupted. Thus scw and tld are necessary for achieving full levels of tinman induction, whereas dpp is obligatory for this event. In addition, unlike dpp mutants, mutants for scw or tld form some residual visceral mesoderm. However, heart formation is more sensitive to the activities of scw and tld and is disrupted to a similar extent as in dpp mutants. schnurri is also necessary for tinman induction in the dorsal mesoderm. The dorsal tinman domain is clearly reduced, as compared to wild-type embryos, although the levels of Tinman mRNA are close to normal. Therefore, shn may be required to enhance dpp signaling during tin induction, but significant levels of tin activation can still occur in the absence of its activity (Yin, 1998).

tinman is essential for dorsal vessel (heart) formation and is structurally and functionally conserved in vertebrates. In the mature embryonic dorsal vessel, tinman is expressed in four of the six pairs of cardioblasts in each segment. Evidence that seven-up, which is homologous to the vertebrate COUP-TF transcription factor and is expressed in the non-Tinman-expressing cardioblasts, represses tinman in these cells. Loss of function seven-up mutations derepress tinman expression in these cardioblasts while ectopic expression of seven-up represses tinman in the cardioblasts that normally express tinman. These changes are correlated with alterations in the expression of additional molecular markers for each of these two types of cardioblasts, such as the novel T-box-containing gene Tb66F2 and the potassium channel-encoding gene sur. These observations suggest that seven-up has a role in diversifying cardioblast identities within each segment. The tinman cis sequences that mediate tinman repression by seven-up are described and whether Seven-up can bind these sequences to directly inhibit tinman was examined. It seems that neither Svp isoform is capable of directly and specifically binding to the 3' half of the tinC element that confers segmental repression of tinman. It is still possible that Seven-up could bind the tinC element in combination with one or more other proteins in order to repress tinman, or Seven-up may indirectly repress tinman in svp cardioblasts through the regulation of downstream genes that affect tinman expression (Lo, 2001).

In late embryonic dorsal vessels, ß-Gal expression from ß-Gal is seen in two adjacent pairs of the six bilaterally symmetrical pairs of cardioblasts per segment. At these stages, Tinman is present in only four of the six contiguous pairs of cardioblasts per segment, and double-staining for ß-Gal and Tinman demonstrate that the svp-lacZ-expressing cardioblasts correspond to the two pairs of non-tinman-expressing cardioblasts. In order to extend this observation and better characterize seven-up expression in the entire dorsal vessel and its relationship to tinman expression, the pattern of ß-Gal and Tinman staining during the development of this organ was examined in embryos from the svp-lacZ line AE127. Tinman protein expression during early dorsal vessel development (stage 11) includes all the heart progenitors, whereas in later stages it becomes restricted to the four pairs of cardioblasts per segment and to a subset of pericardical cells referred to as the tin pericardial cells. In AE127 embryos, the earliest detected expression of ß- Gal in the Tinman-expressing heart progenitors is at mid- stage 11 in a small subset of these cells, when it appears that seven-up is simultaneously expressed in at least two heart progenitor cells in each hemisegment. These cells are irregularly arranged and are first seen in the posterior half of the embryo. By early stage 12, a cluster of four strongly ß-Gal-expressing heart progenitors per hemisegment is seen in seven posterior segments of the embryo, with an additional pair of these cells situated caudally to these clusters. This pattern is maintained from stage 13 until stage 15, when strong ß-Gal expression is observed in two adjacent pairs of cardioblasts in seven segments, plus another single pair located immediately posterior to the last double pair of cardioblasts. The ß-Gal and Tinman double-staining of these late stage dorsal vessels clearly shows that the two pairs of seven-up-expressing cardioblasts correspond to the two pairs of cardioblasts per segment that are not expressing Tinman, such that the two patterns of expression are complementary. The late stage cardioblasts that exclusively express either tinman or svp-lacZ are referred to as the tin and svp cardioblasts, respectively. In addition to the svp cardioblasts, there is also strong expression of svp-lacZ at the anterior end of the dorsal vessel in two bilaterally symmetrical masses of cells that will later fuse at the dorsal midline to form the corpus allatum of the ring gland, an endocrine organ of complex origin (Lo, 2001).

Located laterally to each pair of svp cardioblasts per hemisegment is a pair of cells with much weaker expression of svp-lacZ, which do not express Tinman. Based on their lateral position relative to the cardioblasts and since they appear to arise from dividing svp-lacZ heart progenitors, these cells are considered to be pericardial cells. Since they do not express tinman, these are not tin pericardial cells and thus constitute a novel subtype of pericardial cells. Prior to stage 13, all svp-expressing heart progenitors are positive for Tinman protein (Lo, 2001).

Molecular characterization of the seven-up gene has identified two different transcripts, svp1 and svp2, that are derived from this gene. The svp1 cDNA encodes a 543 amino acid protein that is the Drosophila homolog of the vertebrate COUP-TF subfamily of steroid/nuclear hormone receptors and contains a characteristic N-terminal DNA-binding domain and C-terminal ligand-binding domain. The svp2 cDNA encodes a related 746 amino acid protein that is identical in sequence to the svp1-encoded protein until it completely diverges in the middle of the ligand-binding domain. Since the pattern of ß-Gal expression in the AE127 svp-lacZ line does not differentiate between these two transcripts and may not reflect endogenous mRNA patterns in the heart and its progenitors, in situ hybridizations were performed using probes specific for the unique 3' UTR of each cDNA. The svp1 in situ hybridization pattern in the dorsal vessel during its development appears to be identical to the ß-Gal expression observed in AE127 embryos. svp1 message is first detected cytoplasmically in a portion of the heart progenitors in stage 11 embryos, and in later stages the pattern of expression in the non-Tinman-expressing cardioblasts and the corpus allatum is the same as observed with svp-lacZ. However, while the pattern of dorsal vessel cells expressing svp2 during embryogenesis appears to be identical to svp1, the staining is not cytoplasmic but is instead concentrated in one or occasionally two speckles per nucleus of those cells. In a stage 11 embryo, the svp2 transcript is clearly seen as speckles in the nuclei of a subset of heart progenitors. This speckled intranuclear localization is also seen in several other tissues expressing the svp2 transcript, e.g. the dorsal somatic muscles, but not in other tissues such as the CNS, where it is cytoplasmic, indicating that the intracellular localization of the svp2 transcript is tissue-specific. In the dorsal vessel of stage 15 or older embryos, a pair of adjacent speckles is observed in between the four tin cardioblasts of each hemisegment, with each speckle associated with one svp cardioblast. In addition, there is svp2 expression in the corpus allatum, seen as two larger clusters of speckles at the anterior end of the dorsal vessel (Lo, 2001).

These studies have identified a novel function of seven-up in determining one of two major cell fates in the cardioblasts, as defined by expression of Tb66F2 versus tinman, through repression of the alternative fate. As in the developing retina, seven-up is expressed in a subset of cells in the dorsal vessel, specifically, these cells are the double pairs of non-Tinman-expressing cardioblasts in each segment of the dorsal vessel. The possibility that these two cardioblast types are functionally different in the mature embryonic heart is suggested first by the exclusive expression in the tin cardioblasts of the sur gene, which codes for the Drosophila homolog of the sulfonylurea receptor subunit of the vertebrate ATP-sensitive potassium ion channel. While the vertebrate sulfonylurea receptor has no intrinsic potassium ion channel activity, the Drosophila sur gene has additional sequences not present in the vertebrate SUR genes that endow it with this activity. The presence of the sur gene product in tin cardioblasts could conceivably result in a difference in the electrophysiological properties of these cardioblasts relative to the svp cardioblasts, perhaps in the generation, propagation, and/or control of heartbeat in the dorsal vessel, since it is known that potassium ions are required for proper heartbeat function in Drosophila (Lo, 2001).

Two other differences of the svp cardioblasts from the tin cardioblasts have previously been noted in a study of the embryonic dorsal vessel; the present study utilized as a marker the P-lacZ insertion line E2-3-9 that has recently been identified as a svp-lacZ line. The first difference is that the svp cardioblasts are the cardioblasts initially contacted by the alary muscle cells; this is consistent with the alignment of these cells with the alary muscles in mature embryonic dorsal vessels as seen in the study. However, this morphological feature is not visibly disrupted in svp mutant embryos. The second difference is that in larval stages, these cardioblasts still maintain their compact and rounded shape while the other cardioblasts become larger and flattened. It has been speculated that these svp cardioblasts may be involved in the formation of the ostia -- segmentally repeated openings present in the larval and adult heart that have a valve-like function in allowing the inflow of hemolymph into the heart during diastole. These differences between the tin and svp cardioblasts suggest that the proper specification of these two different cardioblast cell fates during embryogenesis may be crucial for correct dorsal vessel function (Lo, 2001).

In Drosophila, trunk visceral mesoderm, a derivative of dorsal mesoderm, gives rise to circular visceral muscles. It has been demonstrated that the trunk visceral mesoderm parasegment is subdivided into at least two domains by connectin expression, which is regulated by Hedgehog and Wingless emanating from the ectoderm. These findings have been extended by examining a greater number of visceral mesodermal genes, including hedgehog and branchless. Each visceral mesodermal parasegment appears to be divided in the A/P axis into five or six regions, based on differences in expression patterns of these genes. Ectodermal Hedgehog and Wingless differentially regulate the expression of these metameric targets in trunk visceral mesoderm. hedgehog expression in trunk visceral mesoderm is responsible for maintaining its own expression and con expression. hedgehog expressed in visceral mesoderm parasegment 3 may also be required for normal decapentaplegic expression in this region and normal gastric caecum development. branchless expressed in each trunk visceral mesodermal parasegment serves as a guide for the initial budding of tracheal visceral branches. The metameric pattern of trunk visceral mesoderm, organized in response to ectodermal instructive signals, is thus maintained at a later time via autoregulation, is required for midgut morphogenesis and exerts a feedback effect on trachea and ectodermal derivatives (Hosono, 2003).

Metameric RNA expression of bnl, which encodes a ligand for Breathless FGF receptor, is first observed as 12 patches at mid stage 11. bnl RNA expression becomes homogeneous and then diminished during stage 12. tin is a homeobox gene that is required for dorsal mesodermal development. At early stage 10, tin is expressed throughout the dorsal mesoderm from which VM is derived. Metameric Tin expression becomes evident by early stage 11. Tin expression decreases during stage 12. Expression of bap, another homeobox gene required for VM development, can be monitored by bap 4.5#230; (bap-lacZ). Staining for Tin and bap-lacZ or bnl RNA indicates that tin, bap and bnl are co-expressed in VM-PS3-12 during stage 11; in VM-PS2, only bnl is expressed. Stage 11-12 VM also stains for Tin and VM-hh-lacZ. Tin and VM-hh-lacZ expression partially overlaps. VM-hh expression in the anterior terminal region of VM-PSs indicates that each tin/bnl/bap trio expression domain straddles the VM-PS boundary (Hosono, 2003).

In summary, VM-PSs in thorax and abdomen, respectively, are subdivided into five or six regions with respect to differential expression of VM-metameric genes at stages 11-12. Detailed analysis of VM-hh, bnl, tin and bap expression in addition to con indicates that trunk visceral mesodermal genes are classified into three distinct groups -- tin/bnl/bap, VM-hh and con -- and each VM-PS is subdivided into five or six regions, which become apparent during mid stage 11 to stage 12 (Hosono, 2003).

VM is presently considered to develop in two steps under the control of ectodermal Hh and Wg signals. First, by stage 10 (when four mesodermal primordia have become specified), VM competent or bap expression regions are promoted by hh but repressed by wg, via a direct targetor, slp. The second surge of hh and wg activity at stages 10-11 is responsible for subdividing VM-PSs into two regions: con positive and negative. These results indicate that the expression of four other VM-metameric genes, hh, tin, bnl and bap, is also regulated by the second surge of hh and wg activity at stages 10-11 (Hosono, 2003).

In view of morphological changes in a VM competent region and consideration of these findings on VM gene regulation, the following model for VM-PS cell specification is proposed. At stage 10 to early stage 11, anterior terminal cells of VM-PSs are presumed to be situated near an ectodermal AP border, where they are capable of continuously receiving Wg and Hh signals, and Wg confers competence on these cells to express tin/bnl/bap. Wg and Hh are responsible for inducing VM-hh, and Hh, for con expression. In the anterior-most cells, con expression is reduced, which would be expected in view of repression by high Wg signal. The different thresholds of hh for con and VM-hh expression may explain why the con area expands more posteriorly compared with that of VM-hh. Posterior terminal VM cells, when formed, are situated far from Wg expressed on the ectodermal PS border. But as they migrate posteriorly and close to the posteriorly neighboring AP border by early stage 11, they become capable of receiving Wg and acquire competence to express tin/bnl/bap. Thus, the tin/bnl/bap domain would appear regulated by spatially and temporally distinct Wg signals. The two-step induction of tin/bnl/bap expression is supported by experiments using the wg^ts mutant, where, either posterior or anterior expression within one patch can be differentially turned off. Indeed, a stepwise activation of tin/bnl expression is seen in VM-PSs around stage 11. tin and bnl metameric expression became apparent almost simultaneously at mid-stage 11, and preliminary experiments have shown that neither tin nor bnl misexpression can induce the ectopic expression of any other metameric genes examined here. Thus, tin and bnl expression might be initiated in a mutually independent manner (Hosono, 2003).

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).

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).

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