Gene name - tinman
Synonyms - msh-2, NK-4
Cytological map position - 93E1-3
Function - transcription factor
Symbol - tin
Genetic map position - 3-
Classification - homeodomain - NK-2 class
Cellular location - nuclear
|Recent literature||Asadzadeh, J., Neligan, N., Kramer, S.G. and Labrador, J.P. (2016). Tinman regulates NetrinB in the cardioblasts of the Drosophila dorsal vessel. PLoS One 11: e0148526. PubMed ID: 26840059
Morphogenesis of the Drosophila dorsal vessel (DV) shares similarities with that of the vertebrate heart. Precursors line up at both sides of the embryo, migrate towards the midline and fuse to form a tubular structure. Guidance receptors and their ligands have been implicated in this process in vertebrates and invertebrates, as have been a series of evolutionarily conserved cardiogenic transcriptional regulators including Tinman, the Drosophila homolog of the transcription factor Nkx-2.5. NetrinB (NetB), a repulsive ligand for the Unc-5 receptor is required to preserve the dorsal vessel hollow. It localizes to the luminal space of the dorsal vessel but its source and its regulation is unknown. Using genetics together with in situ hybridization with single cell resolution, this study shows how tin is required for NetrinB expression in cardioblasts during DV tubulogenesis and is sufficient to promote NetB transcription ectopically. The study further identifies a dorsal vessel-specific NetB enhancer and shows that it is also regulated by tin in a similar fashion to NetB.
|Howard, A. M., Milner, H., Hupp, M., Willett, C., Palermino-Rowland, K. and Nowak, S. J. (2020). Akirin is critical for early tinman induction and subsequent formation of the heart in Drosophila melanogaster. Dev Biol. PubMed ID: 32950464
The regulation of formation of the Drosophila heart by the Nkx 2.5 homologue Tinman is a key event during embryonic development. This study identified the highly conserved transcription cofactor Akirin as a key factor in the earliest induction of tinman by the Twist transcription factor. akirin mutant embryos display a variety of morphological defects in the heart, including abnormal spacing between rows of aortic cells and abnormal patterning of the aortic outflow tract. akirin mutant embryos have a greatly reduced level of tinman transcripts, together with a reduction of Tinman protein in the earliest stages of cardiac patterning. Further, akirin mutants have reduced numbers of Tinman-positive cardiomyoblasts, concomitant with disrupted patterning and organization of the heart. Finally, despite the apparent formation of the heart in akirin mutants, these mutant hearts exhibit fewer coordinated contractions in akirin mutants compared with wild-type hearts. These results indicate that Akirin is crucial for the first induction of tinman by the Twist transcription factor, and that the success of the cardiac patterning program is highly dependent upon establishing the proper level of tinman at the earliest steps of the cardiac developmental pathway.
tinman is expressed in the mesoderm and is involved in the formation of the heart and dorsal vascular musculature. Oz-like, tinman mutants do not develop a heart. Two questions: first, what chain of chemical events leads to the expression of tinman in the heart, and second, what is tinman's role in heart differentiation?
With regard to the first, the heart (dorsal vessel) is derived from mesoderm, formed as the ventral midsection of the blastoderm undergoes involution. This is one of several morphogenetic tissue movements taking place during the process of gastrulation.
The gene twist is necessary for mesoderm formation. Consequently, tinman is not expressed in twist mutants. twist mutants also exhibit a defective ventral furrow [Image]. Therefore, through the action of twist and its partner in gastrulation, snail, mesoderm comes to occupy the dorsal interior of the embryo, and tinman is induced. But twist is not the sole activator of tinman, and here things get pleasantly complicated.
After gastrulation tinman expression diminishes in the ventral portion of the mesoderm, but it persists and is even enhanced in dorsal mesodermal cells. The part of the mesoderm that will ultimately develop into the heart, the dorsal mesoderm, comes to lie under the dorsal ectoderm. Decapentaplegic is required for the maintenance and enhancement of tinman in these tissues that become the precursors of the fly's heart (Frasch, 1995). even-skipped, another gene also expressed in segmented clusters of pericardial cells from stage 11 onwards, is absent in tin mutants, suggesting that the precursors of pericardial cells fail to become specified (Azpiazu, 1993). Thus tinman expression and heart development is dependent not only on twist and the process of invagination, but also on induction signals from overlying ectoderm once gastrulation is complete.
A functional dissection has been carried out of a tinman enhancer that mediates the Dpp response. Mesoderm-specific induction of tinman requires the binding of both activators and repressors. Screens for binding factors yielded Tinman itself and the Smad4 homolog Medea. The binding and synergistic activities of Smad and Tinman proteins are critical for mesodermal tinman induction, whereas repressor binding sites prevent induction in the dorsal ectoderm and amnioserosa. Thus, integration of positive and negative regulators on enhancers of target genes appears to be an important mechanism in tissue-specific induction by TGF-beta molecules (Xu, 1998).
Functional dissection of the tinman gene has identified a 349-bp enhancer in 3'-flanking regions, tin-D, that is strictly active in dorsal portions of the mesoderm of stage 10-11 embryos. The pattern of lacZ reporter gene expression driven by tin-D closely resembles the dpp-dependent pattern of endogenous tinman expression, suggesting that tin-D functions as a Dpp response element. This notion was further supported by the observation that tin-D reporter gene activity is absent in embryos with a dpp null mutant background. Conversely, upon the ectopic expression of a constitutively active DPP type I receptor, TkvQ-D in the entire mesoderm , tin-D reporter gene expression expands into the ventral mesoderm. The observed changes of tin-D activity when the levels and spatial extents of Dpp signaling are altered closely reflect the changes seen for tinman expression under the same conditions. These observations raise the possibility that the tin-D enhancer is receiving direct inputs from the Dpp signal transduction cascade to activate tinman transcription. In addition to its dependence on dpp, dorsal mesodermal tinman expression requires the activity of tinman itself, as tinman mutant embryos show strongly reduced expression. Correspondingly, full activity of the tin-D enhancer depends on the function of tinman as well. Taken together, these results suggest that Dpp signals and autoregulation by tinman cooperate to induce full levels of tin-D enhancer activity and tinman expression in the dorsal mesoderm (Xu, 1998).
Sequence comparisons between the tin-D elements from Drosophila melanogaster and Drosophila virilis, which displayed identical activities in D. melanogaster embryos, show a high degree of sequence similarity, whereas the similarities in the 5'- and 3'-flanking regions of tin-D elements are considerably lower. The strong sequence conservation between the tin-D enhancers from the two species could reflect the functional conservation of important regulatory sequences. A first inspection of the conserved sequences reveals several candidates for regulatory sites. One of them is a sequence that is present in duplicate, TCAAGTGG, which contains a binding site consensus for homeodomain proteins of the NK family and is identical to previously identified Tinman binding sequences from a heart enhancer of the Drosophila mef2 gene. Tinman protein has specific binding affinity to these sequences in vitro. Another completely conserved sequence is potentially interesting because it contains tandemly repeated CAATGT motifs, with each of the two copies being followed by a stretch of GC-rich sequences at their 3' ends (Xu, 1998).
To define essential regulatory sequences within the tin-D enhancer, a series of derivatives were created with various deletions of the most strongly conserved sequence blocks and their activity was tested in vivo. Three of these fine deletions do not affect lacZ reporter gene expression in transgenic embryos. This indicates that the deleted sequences (nucleotide 16-47, 205-229, and 244-312) either lack any regulatory potential or contain functionally redundant regulatory sequences. In contrast, two other deletions result in a strong reduction of enhancer activity. One of them encompasses the tandemly repeated CAATGT/GC motifs (deltaD3) and causes an almost complete loss of enhancer activity. The other, deltaD6, which deletes 30 bp from the 3' end of tin-D, also yields strongly reduced activity in the dorsal mesoderm. These results show that the subelements D3 and D6 contain important regulatory sequences for the induction of tinman in the dorsal mesoderm and thus are candidates for target sites of the Dpp signaling cascade (Xu, 1998).
To test whether the putative tinman binding sites play roles in autoregulation, the activity of a tin-D derivative, tin-D-deltaD1, in which both of these sites were deleted (nucleotides 1-13 and 197-203). Deletion of these sites provoked two interesting effects. The first is a significant reduction of lacZ reporter gene expression in the mesoderm, which indicates that tinman autoregulation is required to achieve full levels of dorsal mesodermal tinman induction through these sequences. A second, more unexpected effect is observed in the ectoderm. Specifically, embryos carrying tin-D-deltaD1 show strong ectopic reporter gene expression in the dorsal ectoderm, which corresponds to the areas of dpp expression at this stage of development . Accordingly, in a dpp mutant background, both the ectodermal and the residual mesodermal activities of this mutant element are absent. These results show that upon deletion of the tinman binding sites, tin-D is still able to respond to dpp, but its response is essentially switched from the target tissue to the signaling tissue. Therefore, it is concluded that in the normal situation, Tinman binding to these sites is required in an autoregulatory fashion for full induction of tinman by the Dpp signals in the dorsal mesoderm. In addition, the Tinman binding sites appear to overlap with binding sites for an unknown repressor that normally prevents induction of tinman in the dorsal ectoderm, and these two mechanisms together apparently ensure the mesoderm-specific response to Dpp (Xu, 1998).
In the normal situation, tinman autoregulation appears to be restricted to the mesoderm, presumably because the early, twist-activated phase of tinman expression is mesoderm specific. To test whether tinman is also able to autoregulate in the ectoderm, tinman was expressed ectopically and tin-D reporter gene expression was examined under these conditions. For this purpose, tinman was expressed with the binary UAS/GAL4 system in ectodermal stripes under the control of an engrailed driver. Ectodermally expressed tinman is capable of activating tin-D in the ectoderm. Interestingly, ectodermal tin-D expression is restricted to dorsal portions of the transverse Tinman stripes, thereby demonstrating that tinman autoregulation can occur both in the mesoderm and in the ectoderm, but only in conjunction with Dpp signaling (Xu, 1998).
Because a combination of tin-D1 and tin-D3 sequences is sufficient to reproduce a virtually normal expression pattern in the dorsal mesoderm, these combined sequences would appear to contain DNA sequences that can bind the essential factors involved in this inductive process. To identify some of these factors molecularly the yeast one-hybrid system was used to screen for Drosophila cDNAs encoding proteins that specifically bind to D1 or D3 sequences. With multimeric D3 sequences as a bait, GAL4 AD fusion cDNAs were isolated containing sequences with strong similarities to DPC4/Smad4 proteins, which are effectors of various TGF-beta signaling processes. Of 54 candidate yeast clones, 8 carried these sequences and were derived from the same gene. Subsequent sequence comparisons showed that these cDNAs correspond to the Medea gene. Conceptual translation and sequence alignments with other members of this protein family indicate that the encoded GAL4 fusion proteins contain the complete amino-terminal portion of Medea but lack the carboxy-terminal portion encoded by sequences 3' to a native NotI site. It is conceivable that the screen selected against full-length clones, because previous reports have shown that the carboxyl terminus of Smad proteins has autoinhibitory activities (Xu, 1998).
To locate the DNA-binding domain in the Medea protein, a series of carboxy-terminal truncation and in-frame fusion constructs of Medea cDNAs with GAL4 AD coding sequences were generated, and their binding activities were tested in the yeast system, using (D3)5/lacZ as a reporter gene. The activity of Medea products increases on removal of the MH2 domain in this assay, indicating that the MH2 domain of Smad4 group proteins has an inhibitory effect on DNA binding, similar to the MH2 domain of Mad group proteins. By removing 10 carboxy-terminal amino acids from the MH1 domain, the activity drops to background levels. The MH2 domain does not display any binding activities in this assay. Thus, it appears that the MH1 domain serves as the DNA-binding domain for Medea, as it does for Mad (Xu, 1998).
DNase I footprinting assays with bacterially expressed GST fusion proteins were used to characterize the binding of Tinman and Medea to sequences of the tin-D element and to test whether Mad is also able to bind. Tinman specifically protects the two D1 sequences that contain NK homeodomain binding sites and are required for autoregulation. The MH1 domains of Medea protect three distinct sequences within tin-D. Importantly, one of them (nucleotide 95-127) overlaps with the D3 sequence that is essential and sufficient for tin-D activity and was used for the isolation of Medea. Mad shows binding to several additional sequences. Two other sites that are protected by Mad, but not Medea under the same conditions, correspond to the 3' portion of D3 and adjacent sequences. Therefore, Mad protects most of the sequence stretch between nucleotides 95 and 160, which has D3 at its core, whereas Medea protects only the 5' two-thirds of D3. Taken together, the DNase I protection data reveal a minimum of eight in vitro binding sites for Medea and Mad in the tin-D element, at least four of which are located in the essential elements D3 and D6. Moreover, it appears that Medea and Mad have overlapping, but not identical, binding specificities to tin-D sequences (Xu, 1998).
Gel retardation assays provided additional information on the DNA-binding specificities of Medea and Mad and their binding sites in the tin-D element. Both Medea MH1 and Mad MH1 bind to 32P-labeled D3 probes, and excess of unlabeled D3 DNA can compete for binding. Because D3 contains tandemly repeated CAATGT and GC-rich motifs, a test was carried out to find out which of these two sequence motifs are involved in Medea and Mad binding. Replacement of four GCs in each of the GC-rich motifs by As and Ts renders the mutated D3 sequence unable to compete for Medea and Mad binding to the wild-type D3 sequence. In contrast, in vitro mutagenesis of the CAATGT motifs does not interfere with Medea and Mad binding, as these mutated versions compete equally well as D3 wild-type DNA. These data show that the GC-rich motifs are essential for Mad and Medea binding and likely represent two distinct binding sites for these proteins in the D3 element. In summary, these in vitro DNA binding studies demonstrate that the functionally significant D3 and D6 elements contain at least four GC-rich binding sites for Medea and Mad, although Medea binds with high affinity only to those in D3. In addition, tin-D contains at least four other binding sites for Medea and Mad, all of which include GC-rich stretches (Xu, 1998).
In vitro binding sites of Medea and Mad are essential for the activity of tin-D in vivo. Combinations of single copies of the wild-type and mutated sequences of D3 and D6 similar to the ones used for the in vitro binding assays were tested for their ability to activate reporter gene expression in transgenic embryos. The activity of these sequences was tested in the context of a shortened version of tin-D (tin-D*; nucleotide 1-143 plus Tin binding site 2 and nucleotide 321-349). Tin-D* containing wild-type D3 and D6 sequences produces an almost normal pattern of dorsal mesodermal expression, although expression levels are lower than with the complete tin-D element. In contrast, a version in which both Medea/Mad binding sites in D3 are disrupted by 8 bp exchanges is almost completely inactive in vivo. Similarly, expression is nearly abolished upon disruption of the two Mad binding sites in D6 or of all four Medea/Mad binding sites in D3 and D6. Interestingly, specific disruption of the CAATGT sequences in D3 also results in a complete loss of activity in the dorsal mesoderm. Together, these data demonstrate that each of the pairs of Medea/Mad binding sites in D3 and D6 plays a critical role in the Dpp-induced activity of tin-D in the dorsal mesoderm. Moreover, the CAATGT sequences in D3 appear to be required for the binding of a different factor that is also essential during this process. An interesting difference between tin-D and tin-D* is an ectopic expression in the amnioserosa, which is observed between stage 8 and 11 of embryogenesis. This observation suggests that the region between D3 and D6, which is missing in tin-D*, contains a repressor element for this tissue. The results with mutated versions of tin-D* indicate that the Medea/Mad binding sites in D3 and D6 are necessary for amnioserosa expression, whereas the CAATGT sequences are not required (Xu, 1998).
The absolute requirement for the tandemly repeated CAATGT sequences for the activity of the Dpp response element strongly points to the existence of a second essential coactivator that binds to these sequences. The results with wild-type and mutated versions of the tin-D element predict that this factor is expressed and active in both mesoderm and ectoderm, since disruption of the CAATGT motifs abolishes both mesodermal and ectopic ectodermal induction. The close juxtaposition of these motifs with Smad binding sites in the minimal Dpp response element may suggest that the unknown binding factor also participates in protein-protein interactions with bound Smad proteins. It is interesting to note that this sequence motif is closely related to that of the binding site of Xenopus FAST-1. The forkhead domain protein FAST-1 has been shown to bind to the sequences AAATGT within an activin-response element of the Mix.2 gene and to associate with Smad2 and Smad4 . It is thus conceivable that a related member of the forkhead domain protein family plays a similar role in the tinman Dpp response element, albeit in this case in a complex with DNA-associated Smads (Xu, 1998).
Nk-2 proteins are essential developmental regulators from flies to humans. In Drosophila, the family member tinman is the major regulator of cell fate within the dorsal mesoderm, including heart, visceral, and dorsal somatic muscle. To decipher Tinman's direct regulatory role, a time course of ChIP-on-chip experiments was performed, revealing a more prominent role in somatic muscle specification than previously anticipated. Through the combination of transgenic enhancer-reporter assays, colocalization studies, and phenotypic analyses, two additional factors within this myogenic network were uncovered: by activating eyes absent, Tinman's regulatory network extends beyond developmental stages and tissues where it is expressed; by regulating stat92E expression, Tinman modulates the transcriptional readout of JAK/STAT signaling. This pathway is essential for somatic muscle development in Drosophila and for myotome morphogenesis in zebrafish. Taken together, these data uncover a conserved requirement for JAK/STAT signaling and an important component of the transcriptional network driving myogenesis (Liu, 2009).
This article presents a global map of the genomic regions bound by Tinman during multiple stages of mesoderm development, together with functional studies on the direct regulation and phenotypes of selected target genes. This analysis revealed several important features of Tinman's regulatory network. Although Tinman is primarily associated with its conserved role in heart development, many more target genes involved in somatic muscle development were identified. This implies that a major component of Tinman function is to orchestrate the transcriptional network driving early events in this tissue, including myoblast specification. In this context, Tinman's regulatory network is not only active in the dorsal mesoderm; the results pinpoint multiple nodes by which Tinman's regulatory influence extends to lateral and ventral regions of the embryo. First, Tinman directly regulates a number of identity genes essential for lateral and ventral muscle specification. For example, Tinman targets enhancers of slouch, which specifies two ventral muscle fibers (VT1 and VA3), apterous, which specifies three lateral and two ventral muscle fibers (LT1, LT2, LT3, and VA2, VA3), and ladybird (lbe, lbl), which specifies the lateral muscle fiber, SBM. Second, Tinman influences lateral and ventral muscle formation through the regulation of additional transcriptional cascades. A good example is eya, which is an integral component of the Tinman-regulatory network and is essential for the specification of somatic muscle in dorsal, lateral, and ventral regions of the embryo. Similarly, the data suggest that Tinman also directly regulates D-six4 and pox meso expression, two TFs essential for lateral and ventral somatic muscle development. In this manner, Tinman contributes to the general robustness of muscle specification, regardless of the muscle's position along the dorsal-ventral axis (Liu, 2009).
While examining the eya locus, an enhancer was identifed that fully recapitulates the spatiotemporal expression of eya within the mesoderm. This element is occupied by Tinman in vivo and requires tinman function for activity, indicating that Tinman provides direct input into eya expression via this eya-meso enhancer. Despite the strong dependence of the enhancer on tinman function, the expression of the endogenous eya gene is only marginally reduced in tinman mutant embryos. This result indicates a requirement for additional enhancers to regulate high-levels of eya expression within the mesoderm, one of which is most likely regulated by Twist. The expression of the stat92E gene in mesoderm is regulated in a similar manner. These types of regulatory connections (acting partially redundantly or for fine tuning), are often masked in genetics studies, yet serve as important inputs in generating robust regulatory networks (Liu, 2009).
The molecular nature of the ChIP-on-chip approach also uncovered a link between JAK/STAT signaling and muscle development, which was most likely masked in genetics studies due to the pleiotropic function of this pathway. Given the diverse cellular responses to this signaling cascade, including proliferation, apoptosis, and differentiation, the response of Stat activation in the context of myoblasts is currently not clear. A recent study on stat1 in C2C12 cells, a tissue culture model for myogenesis, suggests a potential role in proliferation. While this could partially explain the Drosophila muscle phenotype, it does not readily explain the defects in myotome boundary formation observed in zebrafish (Liu, 2009).
Although there are clear parallels between the role of eya-six genes and the JAK/stat pathway between flies and vertebrates, it is interesting to note that the positions of these genes within the overall myogenic network have diverged significantly. In vertebrates, Pax-Eya-Six act at the top of the transcriptional hierarchy, and are thus involved in the initiation of the myogenic network. In contrast, this regulatory module appears to function further down in the transcriptional hierarchy in flies. As a consequence, the upstream regulators of Eya-Six expression are not conserved. Similarly, as Nkx2 genes are not expressed in somites, stat5.1 cannot be regulated by these TFs in vertebrates, but is rather more likely to be regulated by members of the myogenic bHLH proteins, such as Myf5. Therefore, while the requirement of these key regulators is conserved, the wiring of these nodes within the overall network is highly diverged (Liu, 2009).
In summary, the systematic nature of this approach has revealed important regulators of myogenesis and partially redundant regulatory connections, both of which are often very difficult to uncover using standard genetic approaches (Liu, 2009).
Intercellular signal transduction pathways regulate the NK-2 family of transcription factors in a conserved gene regulatory network that directs cardiogenesis in both flies and mammals. The Drosophila NK-2 protein Tinman (Tin) was recently shown to regulate Stat92E, the JAK/Stat pathway effector, in the developing mesoderm. To understand whether the JAK/Stat pathway also regulates cardiogenesis, a systematic characterization was performed of JAK/Stat signaling during mesoderm development. Drosophila embryos with mutations in the JAK/Stat ligand upd or in Stat92E have non-functional hearts with luminal defects and inappropriate cell aggregations. Using strong Stat92E loss-of-function alleles, this study shows that the JAK/Stat pathway regulates tin expression prior to heart precursor cell diversification. tin expression can be subdivided into four phases and, in Stat92E mutant embryos, the broad phase 2 expression pattern in the dorsal mesoderm does not restrict to the constrained phase 3 pattern. These embryos also have an expanded pericardial cell domain. The E(spl)-C gene HLHm5 is shown to be expressed in a pattern complementary to tin during phase 3, and this expression is JAK/Stat dependent. In addition, E(spl)-C mutant embryos phenocopy the cardiac defects of Stat92E embryos. Mechanistically, JAK/Stat signals activate E(spl)-C genes to restrict Tin expression and the subsequent expression of the T-box transcription factor H15 to direct heart precursor diversification. This study is the first to characterize a role for the JAK/Stat pathway during cardiogenesis and identifies an autoregulatory circuit in which tin limits its own expression domain (Johnson, 2011).
tin expression can be divided into four distinct spatial-temporal phases. Phase 1 tin expression initiates after gastrulation during which Twist (Twi) activates pan-mesodermal tin expression via the enhancer tinB. Phase 2 begins after FGF-mediated mesoderm spreading in which Dpp signals produced by the dorsal ectoderm maintain tin expression throughout the dorsal mesoderm via a second enhancer, tinD. It is during phase 2 that Tin specifies the major dorsal mesoderm derivatives. Phase 3 initiates after dorsal mesoderm progenitor specification and is characterized by a pronounced restriction of tin to the cardiac and visceral muscle progenitors. Upd and Upd2 are expressed in the ventral ectoderm during the transition from phase 2 to phase 3 expression. Phase 4 initiates after precursor specification and is characterized by further restriction of tin to the cardiac precursors that give rise to the contractile cardiomyocytes and the noncontractile pericardial nephrocytes. Phase 4 expression further directs heart cell diversification and maturation and is dependent on a third enhancer element, tinC (Johnson, 2011 and references therein).
To test the hypothesis that the JAK/Stat pathway functions in the cardiac-specific gene regulatory network, a systematic characterization was performed of JAK/Stat signaling during mesoderm development. The JAK/Stat pathway regulates final cardiac morphology as well as heart precursor diversification. Stat92E loss-of-function analysis identified a discrete function for the JAK/Stat pathway in restricting tin during the transition from phase 2 to phase 3 expression. In addition, Stat92E embryos have an expanded pericardial cell domain arguing that the JAK/Stat pathway regulates tin to ensure proper heart precursor diversification. Mechanistically, it was found that the E(spl)-C gene HLHm5 is expressed in a complementary pattern to tin during phase 3 expression and that the JAK/Stat pathway activates HLHm5 expression in the dorsal mesoderm. The E(spl)-C genes in turn repress twi expression to preserve cardiac morphology and restrict tin and H15 expression to direct heart precursor diversification. These findings provide the first evidence of a role for the JAK/Stat pathway in cardiogenesis and identify a novel tin autoinhibitory circuit involving Stat92E and E(spl)-C (Johnson, 2011).
Stat92E is a direct Tin target gene during phase 2 expression; however, Stat92E is expressed in segmented stripes at this stage whereas tin is expressed throughout the dorsal mesoderm. In addition, embryos lacking only the maternal contribution of Stat92E have mesoderm patterning defects. Tin-regulated Stat92E zygotic transcription is therefore insufficient to coordinate mesoderm development. These data suggest that maternally contributed Stat92E is activated in response to segmented Upd and Upd2 activity, binds the Stat92E locus and co-activates Stat92E zygotic transcription with Tin. In addition, ChIP-chip experiments identified Stat92E binding activity and a conserved Stat92E consensus binding sites (SCBS) within the Tin-responsive Stat92E mesoderm enhancer. It is concluded that Stat92E and tin co-regulate a brief, spatially restricted JAK/Stat signaling event that patterns the mesoderm (Johnson, 2011).
Phase 3 tin expression promotes cell-type diversification and differentiation within the dorsal mesoderm and is indirectly activated by Wg via the T-box transcription factors in the Dorsocross complex and the GATA factor Pannier. A key finding of this study is that the JAK/Stat pathway activates the transcriptional repressor HLHm5 in the dorsal mesoderm to establish phase 3 tin expression. Because the HLHm5 cis-regulatory region lacks a conserved SCBS, it is predicted that Stat92E regulates HLHm5 expression through a non-consensus binding site. Alternatively, Stat92E acts at long distances to regulate gene expression. The SCBSs in E(spl)-C could be a platform from which Stat92E regulates multiple E(spl)-C genes that, in turn, regulate HLHm5 expression. In either event, Stat92E-mediated activation of E(spl)-C genes restricts tin in the dorsal mesoderm to establish phase 3 expression. Tin, therefore, establishes an autoinhibitory circuit by activating its own repressors in the JAK/Stat pathway and in E(spl)-C (Johnson, 2011).
Both Stat92E and Df(3R)Esplδmδ-m6 embryos show an increased number of Tin+ pericardial cells and an expanded H15 expression domain. Misexpressing mid or H15 in the mesoderm expands the number of Tin+ cells in the dorsal vessel and embryos misexpressing mid show a phenotype strikingly similar to Stat92E and E(spl) embryos. As mid, and presumably H15, are positively regulated by Tin during St11/12, unrestricted tin expression in Stat92E or Df(3R)Esplδmδ-m6 embryos expands the H15 expression domain. Ectopic H15 then specifies supernumerary Tin+ pericardial cells. Because mid expression is not affected in Stat92E embryos, the expression of mid and H15 must be controlled by distinct mechanisms and might have non-redundant functions (Johnson, 2011).
Although the Twi target genes directing mesoderm development and subdivision have been studied in detail, the molecular mechanism that restricts twi expression after gastrulation remains unclear. One regulator of twi is the Notch signaling pathway, which acts through E(spl)-C genes to restrict twi expression. However, Notch signaling appears to be active throughout the mesoderm after gastrulation. This study suggests that segmented JAK/Stat signaling activity differentially upregulates E(spl)-C gene expression in concert with Notch to produce periodic twi expression in the mesoderm. In addition, pan-mesodermal twi expression causes cardiac defects independently of cell fate specification, suggesting that the cardiac morphology defects in Stat92E embryos are due to dysregulated twi expression. These results highlight a previously unrecognized role for the JAK/Stat pathway and Twi in regulating cardiogenesis (Johnson, 2011).
Pericardial cell hyperplasia without a concomitant loss of cardioblasts has been reported for dpp hypomorphic embryos and lame duck (lmd) embryos. A late Dpp signal, which occurs after the Dpp signal that regulates phase 2 tin expression, instructs the Gli-like transcription factor Lmd to repress Tin expression in fusion competent myoblasts (FCMs). Tin expression appears to expand into the FCM domain in Stat92E embryos; however, the closest Stat92E chromatin binding domain is over 120 kb distal to the lmd transcriptional start site. This study highlights the possibility that sequential JAK/Stat and then Dpp signals regulate Lmd function to direct heart precursor diversification (Johnson, 2011).
In vertebrates, skeletal myogenesis initiates with the periodic specification of somites in the presomitic mesoderm. Cyclical expression of hairy1 in the chick, the hairy- and E(spl)-related family (Her) in zebrafish, and the orthologous Hes family in mice are under the control of Notch-Delta signaling. Loss of her1 and her7 alters the periodicity with which somite boundaries are specified in fish, and artificially stabilizing Hes7 causes somites to fuse in the mouse. Thus, mesoderm segmentation is governed by Notch-Delta regulation of the E(spl)-C genes in both insects and vertebrates indicating that the two processes share molecular homology. A cell culture model of somitogenesis shows that oscillating Hes1 expression is dependent on Stat activity. This study supports the exciting possibility that JAK/Stat signaling and E(spl)-C form a conserved developmental cassette directing mesoderm segmentation throughout Metazoa (Johnson, 2011).
The 93D/E homeobox gene cluster is composed of two Drosophila genes, lady-bird-late (previously named nkch4) and lady-bird-early, when taken together with tinman/NK4, bagpipe/NK3, S59/NK1 and 93Ba1 (Jagla, 1994).
Bases in 5' UTR - 289
Bases in 3' UTR - 145
Tinman is a homeodomain protein with several CAX repeats (Bodmer, 1990 and Kim, 1989).
date revised: 30 August 98
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