optomotor-blind-related-gene-1: Biological Overview | References
Gene name - optomotor-blind-related-gene-1
Cytological map position - 7E9-7E10
Function - transcription factor
Symbol - org-1
FlyBase ID: FBgn0021767
Genetic map position - chrX:8341562-8349042
Classification - T-box DNA binding domain
Cellular location - nuclear
Members of the T-Box gene family of transcription factors are important players in regulatory circuits that generate myogenic and cardiogenic lineage diversities in vertebrates. This study shows that during somatic myogenesis in Drosophila, the single ortholog of vertebrate Tbx1, optomotor-blind-related-gene-1 (org-1), is expressed in a small subset of muscle progenitors, founder cells and adult muscle precursors, where it overlaps with the products of the muscle identity genes ladybird (lb) and slouch (slou). In addition, org-1 is expressed in the lineage of the heart-associated alary muscles. org-1 null mutant embryos lack Lb and Slou expression within the muscle lineages that normally co-express org-1. As a consequence, the respective muscle fibers and adult muscle precursors are either severely malformed or missing, as are the alary muscles. To address the mechanisms that mediate these regulatory interactions between Org-1, Lb and Slou, distinct enhancers associated with somatic muscle expression of lb and slou. These lineage- and stage-specific cis-regulatory modules (CRMs) bind Org-1 in vivo, respond to org-1 genetically and require T-box domain binding sites for their activation. In summary, it is proposed that org-1 is a common and direct upstream regulator of slou and lb in the developmental pathway of these two neighboring muscle lineages. Cross-repression between slou and lb and combinatorial activation of lineage-specific targets by Org-1-Slou and Org-1-Lb, respectively, then leads to the distinction between the two lineages. These findings provide new insights into the regulatory circuits that control the proper pattering of the larval somatic musculature in Drosophila (Schaub, 2012).
The musculature and the central nervous system are two examples of tissues featuring a high diversity of cell types. In each hemisegment of the Drosophila embryo, about 30 distinct body wall muscles and six adult muscle precursors are formed and in the CNS the number of neuronal and glial cell types generated is an order of magnitude higher. Currently, only a limited picture is available of the regulatory processes that generate these great cellular diversities, but several steps of muscular and neuronal development in Drosophila known to be accomplished by closely related processes. For example, the earliest progenitor cells, termed muscle progenitors and neuroblasts, respectively, seem to be defined by intersecting anterior-posterior and dorsoventral upstream regulators. In both tissues, these progenitors are preceded by larger clusters of promuscular or proneural cells, respectively, that are characterized by their expression of proneural genes from the Achaete-Scute complex. Lateral inhibition mediated by Delta/Notch signals singles out individual progenitor cells from these clusters. In the case of the muscle progenitors, receptor tyrosine kinase (RTK) signaling, particularly via epidermal growth factor (EGF)- and fibroblast growth factor (FGF)-receptors (Egfr and Htl, respectively), is required as a positive input to antagonize Notch. An additional similarity is the occurrence of asymmetric cell divisions that involve the unequal segregation of the Notch-inhibitor Numb. In the case of muscle progenitors, these asymmetric divisions typically generate two distinct muscle founder cells that go on to form two different larval muscle fibers. In some instances, one sibling of a muscle founder cell gives rise to certain heart precursors or to a stem cell-like adult muscle progenitor that will contribute to the adult body wall musculature. Subsequently, muscle founder myoblasts fuse with surrounding fusion-competent myoblasts to form distinct body wall muscle fibers. During both muscle and neuronal development, the increasing diversity of the newly generated cells is reflected in the distinct combinations of transcription factors expressed in them. Several of these factors are known to have key roles in defining the particular cell identities and have been called muscle identity factors (Schaub, 2012).
Although the broad picture of these diversification events is known, there are major gaps in knowledge with regard to the specific design of the regulatory networks involved. In the case of Drosophila muscle development, a few lineages of muscle progenitors have been characterized in some detail. The first are those marked by the expression of the identity factor Even-skipped (Eve, a homeodomain protein) and have been best characterized with regard to the essential early-acting inputs. These particular muscle progenitors arise in the dorsal portion of the mesoderm and give rise to somatic muscle fibers as well as to specific cells of the dorsal vessel. One of the two adjacent muscle progenitors marked by Eve generates the founder of muscle 1 (DA1), which requires eve function, whereas the other generates the founder of muscle 10 (DO2). The activation of eve in these muscle progenitors requires combinatorial signals from ectodermal Decapentaplegic (Dpp) and Wingless (Wg), the mesodermal competence factor Tinman, which itself is downstream of Dpp, as well as RTK signals via Egfr and Htl. All these combinatorial signaling and transcription factor inputs are directly integrated at the level of a mesoderm-specific enhancer element at the eve locus (Schaub, 2012).
The step-wise specification of muscle identities has been addressed for muscle founders expressing the identity factor Collier (Col, a COE transcription factor; Knot -- FlyBase). In abdominal segments, Col is expressed in the two adjacent dorsolateral muscle progenitors of muscles 3 (DA3) and 20 (DO5) as well as 18 (DT1) and 19 (DO4) and the corresponding promuscular clusters. The expression is only maintained in the founder and precursor of muscle 3/DA3, which requires Col for its development. This dynamic expression is reflected in the activities of at least two cis-regulatory modules (CRMs) of col. An intronic CRM activates expression within both Col+ promuscular clusters and the progenitors singled out from them, whereas an upstream CRM activates and maintains expression specifically in the muscle founder 3/DA3 and its muscle. The early-acting intronic CRM responds to positional and mesodermal inputs, which might involve binding by Twist and Tinman, whereas activation of the later-acting CRM in the M3/DA3 founder relies on the combinatorial binding of autoregulatory Col, Nautilus (Nau, also known as MyoD), and the Hox factors Antp and Ubx. In this manner, the early patterning and tissue-specific inputs are integrated with the axial inputs of the Hox genes that modulate the muscle pattern and fiber size in different body parts (Schaub, 2012).
The muscle lineages that are the focus of the present study are positioned laterally in the somatic mesoderm and are marked by the expression of the homeodomain factors Slouch (Slou) and Ladybird (Lb), which function as identity genes in these lineages [lb refers herein to the paralogous ladybird early (lbe) and ladybird late (lbl) genes]. Within this area of interest, Slou is expressed in the progenitor and sibling founders of muscles 5 (LO1) and 25 (VT1) and is crucial for the formation of these muscles. Lbe is expressed directly adjacent to Slou in a promuscular cluster and then two muscle progenitors. One of these progenitors forms muscle 8 (SBM) and a lateral adult muscle precursor (lAMP), which continue to express Lb, whereas the other forms a second Lb+ lAMP and a sibling that probably undergoes apoptosis. Genetic and genomic analyses indicate that Lb has important functions in the specification and/or differentiation of muscle 8 (Schaub, 2012).
The expression of slou and lbe appears to be controlled by an overlapping set of upstream regulators, because their mutually exclusive expression in the neighboring progenitors of muscles 5/25 and muscle 8/lAMP requires cross-repression by their respective gene products. However, the nature of these shared regulatory inputs has been unknown. The present study identified org-1, a Drosophila Tbx1 ortholog that had not been characterized extensively prior to this work (Porsch, 1998; Lee, 2003), as a key activator of both slou and lbe in this pathway. org-1 expression in the mesodermal areas and progenitors that will form muscles 5, 25, 8 and the Lb+ lAMPs precedes that of slou and lbe, and the activation of these identity genes requires org-1 activity. Consequently, the development of these muscles and the lAMPs is disrupted in org-1 mutant embryos. The activation of slou and lbe by org-1 requires T-box binding motifs in their respective founder-specific enhancer elements and show in vivo occupancy of these elements by Org-1. Hence, slou and lbe appear to be direct target genes of org-1 in the developmental pathway of these neighboring muscle lineages. In addition, org-1 is expressed in the progenitor of the alary muscles and its function is needed for the development of these muscles, which form segmental anchors of the dorsal vessel (Schaub, 2012).
Analysis of the expression and function of org-1 in somatic muscle development has established this gene as a new and crucial representative of muscle identity genes in Drosophila. The data have provided new insights into developmental controls in two well-defined muscle lineages and somatic muscle development in general. These lineages include one dependent on the homeobox gene slou, which gives rise to muscles M5 and M25 (also known as 'cluster I'), and another dependent on the lb homeobox genes, which gives rise to the segment border muscle M8 and lateral adult muscle precursors (lAMPs). They arise from promuscular clusters and muscle progenitors abutting each other in the ventrolateral somatic mesoderm. Until now, it was assumed that slou and lb are positioned at the top of the regulatory hierarchy set up within each of these lineages, as their expression and function is already detected at the progenitor cell stage prior to asymmetric divisions into different founder cells. As in the model proposed for the even-skipped-expressing lineage in the dorsal somatic mesoderm, the progenitor-specific expression of slou and lb was assumed to be determined directly by the antagonistic actions of various more broadly active activators and negative influences through lateral inhibition via Notch. Based on genetic assays using the expression of slou and lb, and the formation of the respective muscles as outputs, good candidates for such activators included localized receptor tyrosine kinase (RTK)/Ras/MAPK signals in conjunction with the relatively broadly expressed mesodermal transcription factors Tinman, Six4 (in the case of lb), Twist and Sloppy paired. Ectodermal Wg signals were also found to regulate these Slou+ and Lb+ lineages, although at least in the case of slou these apparently act indirectly by upregulating twi and slp expression (Schaub, 2012).
The current findings have uncovered an additional layer of regulation upstream of slou and lb within the M5/M25 and M8/lAMP lineages that involves org-1 (see Org-1 is a direct regulator of ladybird and slouch and is required for cell fate specification and differentiation in distinct lateral muscle cell lineages). The initial expression of org-1 occurs in segmented areas of the lateral somatic mesoderm, from which the org-1+ M5/M25 and M8/lAMP progenitors emerge. These areas probably delineate the two abutting promuscular clusters from which these progenitors are singled out (as well as a third one dorsolaterally, from which the progenitor and founder of the alary muscle is formed). Although this early expression of org-1 is not required for progenitor formation per se (based upon the normal pattern of org-1:HN39-lacZ in progenitors of org-1 mutants), org-1 is crucial either during this phase or during early phases of progenitor formation for activating slou and lb in the respective progenitors. Hence, it is proposed that the previously identified regulators of slou and lb, including Notch, RTKs, tin, wg, twi and slp, act predominantly in establishing progenitor-specific expression of org-1, which in turn activates slou and lb. However, a plausible alternative to this linear model of regulation would be a feed-forward model, in which some of the above upstream regulators are re-employed during the second step to activate slou and lb together with mandatory Org-1. Whether org-1 activates slou or lb in any given progenitor would depend on the outcome of the previously reported mutual inhibition between slou and lb. It is thought unlikely that the outcome of this process is completely random; instead, a mechanism is favored involving an initial bias towards one or the other. Such biases could, for example, arise through slight differences in the spatial activities of the EGF and FGF receptors (Egfr and Htl), or of transcription factors such as Six4, coupled with differential responses of slou and lb to these regulators. Regardless of the specific mechanism, this principle of joint activation followed by mutual repression allows for the differential specification of directly neighboring cells, in this case of P5/25 and P8/lAMP, that are under the influence of common upstream regulators (Schaub, 2012).
The combination of genetic data and functional enhancer analysis provides convincing evidence that both slou and lb are direct transcriptional targets of Org-1. This conclusion also fits nicely with the observation that a slou enhancer fragment used herein, SK16, is active ectopically in M8 and that Org-1 and the putative Org-1 binding sites are required for its activity not only in M5 and M25, but also in M8. By contrast, an adjacent enhancer fragment, SK10, is not active in M8 and thus reflects the endogenous Org-1-dependent pattern of slou. Presumably unlike SK16, this element still includes lb-dependent repression elements that block its activation by Org-1 in M8 (Schaub, 2012).
The lbl-SBM enhancer (1.36 kb) was identified initially in a bioinformatics approach for sequences near founder cell-expressed genes that are enriched with binding motifs for Twi, Tin, dTCF (Pan - FlyBase; Wg signaling effector), Pointed (RTK signal effector) and Mad (Dpp signaling effector), and also showed increased evolutionary conservation. However, this enhancer had a relatively low score and, furthermore, the Tin and Twi motifs with decent matches to bona fide binding sites for these factors lie outside of a shortened version, lbl-SBMs. Additionally, Smad proteins are not expected to be active in this ventrolateral region. It is possible that this enhancer was picked up with this algorithm largely because of the adjacent high-scoring cardiac enhancer. Nevertheless, some of the remaining binding motifs within lbl-SBMs, e.g. those for dTCF and Pnt, might contribute to the enhancer activity in progenitors and founders together with the essential Org-1 binding sites. The residual activation of the lbl-SBMs enhancer with mutated Org-1 binding motifs in the differentiated M8 shows that the maintenance of enhancer activity at later stages depends on regulators other than Org-1, which agrees with the observation that Org-1 is no longer expressed in M8 during these stages. This maintenance might be regulated by inputs shared with two separate later-acting enhancer elements upstream of lbe (termed LAMPE) and lbl (termed LME). Both of these elements include homeodomain binding motifs that probably mediate autoregulation by Lb, in addition to ETS domain and Mef2-binding motifs, respectively, that also contribute to full activity in M8. In org-1 mutants, the lbl-SBM enhancer is inactive not only early but also late, possibly because Lb protein is not available for autoregulation (Schaub, 2012).
Unlike in slou mutants or embryos with ectopic lb expression, in org-1 mutants the muscles derived from the progenitors of M5/25 and M8/lAMP are not transformed into other muscles with recognizable identities. Instead, they form syncytia with variable shapes that do not appear to be properly attached to tendon cells. This phenotype is explained by the requirement of org-1 for the activation of both slou and lb. Whereas in slou mutants the progenitor of M5/M25 expresses ectopic lb, which transforms it into a second progenitor of M8/lAMP, in org-1 mutants de-repression of lb is not possible as org-1 is also needed for the activation of lb. Hence, neither of the two progenitors is able to express any of these identity genes that would allow them to form identifiable muscle fibers, although they still divide into founder cells that form syncytia. Only the founder of M25 forms a syncytium that resembles M25 to some extent, perhaps because it still expresses an unknown, org-1-independent identity gene that can establish some of the characteristics of M25 in the absence of slou and org-1 function (Schaub, 2012).
Vertebrate Tbx1 is also expressed prominently in mesodermal areas that form muscles, although in this case not in the trunk region but rather in the core mesoderm of the pharyngeal arches that form branchiomeric muscles of the head. Notably, Tbx1 is also expressed in pharyngeal arch core mesoderm derived from anterior portions of the second heart field, which makes prominent contributions to the outflow tract and right ventricle of the heart. All of these muscle and heart tissues are disrupted upon mutation of Tbx1 in the mouse and are also affected in patients suffering from the DiGeorge (or velo-cardio-facial) syndrome, in which mutations and deletions cause haploinsufficiency of TBX1. No obvious connections are seen between Drosophila org-1 function and the cardiogenic roles of vertebrate Tbx1, as org-1-expressing cells do not contribute to the dorsal vessel proper and the org-1+ alary muscles are specialized skeletal muscle fibers rather than genuine cardiac muscles. However, conceivably some of the regulatory interactions upstream or downstream of Tbx1/org-1 during the development of vertebrate head muscles and Drosophila trunk muscles have been conserved. This could, for example, be the case during tongue muscle development, in which the lb ortholog Lbx1 is co-expressed with Tbx1. The identification of larger numbers of upstream regulators and targets of both org-1 and Tbx1 will be required in order to obtain a clearer picture of the evolutionary conservation and divergence of the developmental functions of these genes (Schaub, 2012).
The T-Box family of transcription factors plays fundamental roles in the generation of appropriate spatial and temporal gene expression profiles during cellular differentiation and organogenesis in animals. This study reports that the Drosophila Tbx1 orthologue optomotor-blind-related-gene-1 (org-1) exerts a pivotal function in the diversification of circular visceral muscle founder cell identities in Drosophila. In embryos mutant for org-1, the specification of the midgut musculature per se is not affected, but the differentiating midgut fails to form the anterior and central midgut constrictions and lacks the gastric caeca. It was demonstrate that this phenotype results from the nearly complete loss of the founder cell specific expression domains of several genes known to regulate midgut morphogenesis, including odd-paired (opa), teashirt (tsh), Ultrabithorax (Ubx), decapentaplegic (dpp) and wingless (wg). To address the mechanisms that mediate the regulatory inputs from org-1 towards Ubx, dpp, and wg in these founder cells, known visceral mesoderm specific cis-regulatory-modules (CRMs) of these genes were dissected. The analyses revealed that the activities of the dpp and wg CRMs depend on org-1, the CRMs are bound by Org-1 in vivo and their T-Box binding sites are essential for their activation in the visceral muscle founder cells. It is concluded that Org-1 acts within a well-defined signaling and transcriptional network of the trunk visceral mesoderm as a crucial founder cell-specific competence factor, in concert with the general visceral mesodermal factor Biniou. As such, it directly regulates several key genes involved in the establishment of morphogenetic centers along the anteroposterior axis of the visceral mesoderm, which subsequently organize the formation of midgut constrictions and gastric caeca and thereby determine the morphology of the midgut (Schaub, 2013).
The analysis of org-1 expression and function during visceral mesoderm development defined this gene as a new and essential lineage specific regulator of circular visceral muscle founder cell identities and midgut patterning in Drosophila. The data add new insights into the developmental regulatory mechanisms responsible for the diversification of the circular visceral muscle founder cell lineage and midgut morphogenesis (Schaub, 2013).
The initial expression of org-1 occurs in the segmented trunk visceral mesoderm (TVM), where it is coexpressed with tin, bap, bin and Alk. It has been documented that the induction of tin and bap in the dorsal mesoderm involves the combined binding of Smad proteins (Medea and Mad) and Tin to Dpp-responsive enhancers of the tin and bap genes, whereas the segmental repression of bap is mediated by binding of the sloppy paired (slp) gene product. Genetic analysis of org-1 has shown that org-1 is activated downstream of tin but independently of bap and bin, and that dpp provides the key signals for its induction. This suggests a regulatory mechanism analogous to that of bap, in which the combined binding of Smads and Tin activates a Dpp-responsive org-1 enhancer, whereas Wg activated Slp is required for its mutual segmental repression (Schaub, 2013).
The similarities in the early expression patterns of bap, bin, Alk and org-1 in the trunk visceral mesoderm primordia raise the question of the contribution of org-1 to the early development of the TVM as such. Whereas bap and bin are crucially required for the specification of the trunk visceral mesoderm and visceral musculature, loss of org-1 function, like the loss of Alk, has no obvious impact on the specification of the early TVM. Therefore, it is notable that during the subdivision of the visceral mesoderm primordia into founder and fusion-competent myoblasts (cFCs and FCMs), org-1 expression is extinguished in the FCMs and only sustained in the cFC lineage of the circular visceral musculature. This lineage-specific restriction and maintenance of org-1 expression crucially depends on Jeb mediated Alk/Ras/MAPK signaling and points toward a possible cFC lineage specific function of org-1. The genetic analysis demonstrates that org-1 is not required for cFC specification, but plays a decisive role in the induction of the visceral mesoderm specific expression of patterning genes in the founder cells of the circular musculature. Thus, org-1 is critical for the processes of cell fate diversification that provide individual fields of cells along the anteroposterior axis of the visceral mesoderm with their specific identities (Schaub, 2013).
Proper anteroposterior patterning of the trunk visceral mesoderm and the formation of localized organizer fields are prerequisites for eliciting the morphogenetic events that shape the midgut. The formation of these organizer fields depends on the appropriate spatial expression domains of the homeotic selectors Scr, Antp, Ubx and abd-A, the secreted factors dpp and wg, as well as the zinc finger proteins opa and tsh, which are required for the formation of the midgut constrictions as well as the gastric caeca. The regulatory mechanisms responsible for the establishment of the spatial, temporal and tissue-specific expression patterns of these genes in the TVM are only partially understood. Genetic and molecular analyses with the FoxF gene bin, which is expressed in all trunk visceral mesoderm precursors and their descendents, have demonstrated that bin is a direct upstream regulator of dpp in PS7 and is also required for the expression of wg in PS8 of the TVM. Thus, Bin serves as an essential TVM-specific competence factor in conjunction with the dpp/wg signaling feedback loop. The current findings have defined Org-1 as an additional tissue-specific regulator with an even broader range of downstream patterning genes in the TVM, but with a narrower spatial range of action. org-1 acts specifically within the visceral muscle founder cell lineage as a positive regulator upstream of opa, tsh, Ubx, dpp as well as wg (Schaub, 2013).
This combination of genetic data and functional enhancer analyses provides convincing evidence that both dpp and wg are direct transcriptional targets of Org-1 in the cFCs. Prior dissections of the dpp visceral mesoderm (VM) enhancer had shown that it is also regulated by the direct binding of Ubx, Exd, dTCF (a Wg effector) and Bin, and that minimal synthetic variants that contain only the binding motifs for Ubx, Exd, Bin, and dTCF within conserved sequence contexts (which happen to include the Org-1 motif) are active as VM enhancers. Likewise, the wgXC enhancer fragment integrates Org-1 with the direct regulatory inputs of Abd-A as well as CREB and Smad (Mad/Medea) proteins mediating Dpp signaling (Schaub, 2013).
Org-1 is the first transcription factor known to be required for Ubx expression in PS7 of the visceral musculature. Extensive work on an Ubx visceral mesoderm CRM (UbxRP) indicated that dpp and wg regulate Ubx through indirect autoregulation. Of note, in bin embryos, which also lack visceral mesodermal dpp and wg expression, Ubx is still expressed. Genetic data show that the UbxRP element, while requiring org-1, is not directly regulated by Org-1, since mutation of its four predicted T-Box binding sites did not have any effects. Taking into account that no UbxRP reporter activity was detected in the cFCs at pre-fusion stages, it is suggested that UbxRP represents a late enhancer element and responds to dpp and wg only after they are activated by Org-1 in the founder cells. To clarify whether the regulation of Ubx by Org-1 is direct or indirect, the identification and dissection of a founder cell specific CRM will be required (Schaub, 2013).
tsh and opa were described as homeotic target genes of Antp in PS4-6 (tsh) and PS4-5 (opa) as well as of abd-A in PS8 (tsh) and PS9-12 (opa) of the visceral musculature. The current data show that tsh and opa expression is already activated in the respective cFCs of the visceral parasegments where it requires org-1. The later activation of tsh in PS8 during muscle fusion follows the org-1 dependent founder cell specific initiation of wg in PS8, which acts upstream of tsh. Thus it was conceivable that the regulation of tsh by org-1 is indirect. However, ectopic activation of wg in an org-1 loss of function background is not able to rescue tsh expression and Antp and abd-A expression is not altered upon loss of org-1. These observations suggest that Org-1 acts directly on tsh and opa, e.g., via functional cooperation with Antp and Abd-A, respectively, during the early activation of tsh and opa in the founder cells (Schaub, 2013).
It was reported that the absence of Jeb/Alk signaling causes loss of dpp expression in the founder cells in PS7 of the visceral mesoderm. In light of the current findings that org-1 loss-of-function produces a similar phenotype, and of the previous demonstration that org-1 expression is downstream of Jeb/Alk, this observation could simply be explained by the action of a linear regulatory cascade from Jeb/Alk via org-1 towards dpp. Alternatively, Jeb/Alk may provide additional inputs towards dpp (and other patterning genes) in parallel to org-1, which could explain the slightly stronger phenotype of Alk as compared to org-1 mutations with respect to dpp. A possible candidate for an additional effector of Jeb/Alk signals in this pathway is extradenticle (exd), which is known to be required for normal dpp expression in PS7 of the visceral mesoderm, presumably through direct binding of Exd in a complex with Hox proteins and Homothorax (Hth) to a PS7-specific enhancer element (a derivative of which was used in this study). Like org-1, exd is also needed for the expression of tsh and wg in the visceral mesoderm (Additionally, it represses dpp in PS4-6 through sequences not contained in the minimal PS7 enhancer). It is thought that Exd complexed with Hox proteins and Hth increases the binding preference of these Hox complexes for specific binding sites within visceral mesodermal enhancers of their target genes (Schaub, 2013).
Since exd is expressed in both founder and fusion-competent cells in the visceral mesoderm, it is unlikely that it fulfills its roles in the regulation of dpp, wg, and tsh in the founder cells as a downstream gene of org-1. However, it is known that Exd requires nucleocytoplasmic translocation for it to be functiona and, interestingly, it has been shown that Jeb/Alk signals trigger nuclear localization of Exd specifically in the cFCs of the visceral mesoderm. Because nuclear Exd appears to be hyperphosphorylated as compared to cytoplasmic Exd, nuclear translocation of Exd may be triggered by Alk-mediated phosphorylation. Alternatively, Jeb/Alk signals may induce the expression of hth in the cFCs and Hth could then translocate Exd to the nuclei, as has been shown in other contexts. This would be compatible with the observation that Hth is upregulated in the founder cells in an org-1-independent manner (Schaub, 2013).
The combined data show that Jeb/Alk signals exert at least two parallel inputs towards patterning genes in the cFCs, which are the induction of org-1 and the nuclear translocation of Exd. Taken altogether, a model is suggested in which combinatorial binding of Org-1, nuclear Exd/Hth and the homeotic selector proteins to the corresponding visceral mesoderm specific CRMs is required for the initiation of lineage specific expression of opa, tsh, dpp, Ubx and wg in the founder cells of the respective parasegments. As shown in the examples of dpp (PS7) and wg (PS8), accessory Bin is required for the activation as a general visceral mesodermal competence factor, whereas Dpp and Wg effectors mediate autoregulatory stabilization of their expression (Schaub, 2013).
Extensive work has shown that during somatic muscle development individual founder myoblasts acquire distinct identities, which are adopted by the newly incorporated nuclei upon myoblast fusion, thus leading to the morphological and physiological diversification of the differentiating muscles. It is proposed that the same principle is active during visceral muscle development. In this view, Org-1 acts as a muscle identity factor in both the somatic and visceral mesoderm. In the visceral mesoderm, Org-1 helps diversifying founder cell identities and, after myoblast fusion, their differential identities are transmitted to the respective differentiating circular gut muscles. The activation of downstream targets of this identity factor in the developing muscles leads to the observed morphogenetic differentiation events of the midgut and the establishment of the signaling center in PS7/8 that is also required for Dpp and Wg mediated induction of labial in the endodermal germ layer. As is the case for identity factors in the somatic muscle founders, Org-1 in the visceral mesoderm acts in concert with other, spatially restricted activities such as Hox factors and signaling effectors to achieve region-specific outputs. The main difference is that, in the trunk visceral mesoderm, Org-1 is present in all founder cells whereas in the somatic mesoderm this identity factor (like others) is expressed in a particular subset of founder myoblasts. Thus, in contrast to the somatic mesoderm, the spatial expression of Org-1 does not contribute to its function in visceral muscle diversification and instead, it solely relies on spatially-restricted co-regulators during this process (Schaub, 2013).
The pool of trunk visceral mesodermal fusion-competent cells contributes to the formation of both circular and longitudinal midgut muscles, depending on whether they fuse with resident founder cells of the trunk visceral mesoderm or with founders that migrated in from the caudal visceral mesoderm. The restricted expression of the identity factor Org-1 in the founder myoblasts in the trunk visceral mesoderm and its exclusion from the FCMs represents an elegant mechanism to ensure that the respective patterning events only occur in the developing circular musculature but not in the longitudinal muscle fibers, which extend as multinucleate syncytia throughout the length of the midgut (Schaub, 2013).
The T-box transcription factor Tbx1 and the LIM-homeodomain transcription factor Islet1 are key components in regulatory circuits that generate myogenic and cardiogenic lineage diversity in chordates. This study shows that Optomotor-blind-related-gene-1 (Org-1) and Tup, the Drosophila orthologs of Tbx1 and Islet1, are co-expressed and required for formation of the heart-associated alary muscles (AMs) in the abdomen. The same holds true for lineage-related muscles in the thorax that have not been described previously, which were named thoracic alary-related muscles (TARMs). Lineage analyses identified the progenitor cell for each AM and TARM. Three-dimensional high-resolution analyses indicate that AMs and TARMs connect the exoskeleton to the aorta/heart and to different regions of the midgut, respectively, and surround-specific tracheal branches, pointing to an architectural role in the internal anatomy of the larva. Org-1 controls tup expression in the AM/TARM lineage by direct binding to two regulatory sites within an AM/TARM-specific cis-regulatory module, tupAME. The contributions of Org-1 and Tup to the specification of Drosophila AMs and TARMs provide new insights into the transcriptional control of Drosophila larval muscle diversification and highlight new parallels with gene regulatory networks involved in the specification of cardiopharyngeal mesodermal derivatives in chordates (Boukhatmi, 2014).
The anatomical organization of the Drosophila larval organs is established during embryogenesis. This study show shere that abdominal AMs and their thoracic counterpart, the TARMs, establish physical connections between the exoskeleton and different internal organs, and that TARMs thus represent a novel type of muscle. The control of AM and TARM development by Org-1 and Tup - as orthologs of Tbx1 and Islet1, two key transcription factors of cardiopharyngeal mesoderm development in vertebrates - suggests that these transcription factors were part of an ancestral regulatory kernel that was redeployed several times during evolution to control different embryonic mesoderm derivatives (Boukhatmi, 2014).
Seven pairs of embryonic abdominal AMs connect to the heart and the aorta. Interestingly, pan-mesodermal expression of either Ubx or AbdA, two abdominal Hox proteins, resulted in the formation of three supernumerary pairs of AMs arranged in a segmental pattern in the three thoracic segments. This suggested the existence of thoracic PCs that fail to give rise to muscles in the absence of proper Hox input. Such a scenario accounted for the lack of DA3 muscle in the T1 segment. Alternatively, these thoracic PCs could give rise to previously unrecorded muscles. Both explanations were found to apply. Thoracic Org-1+/Tup+ PCs give rise to a novel type of muscles, which were call TARMs. Only TARMT1 and TARMT2 form during normal development, whereas TARMT3 differentiation is abortive. An extra pair of muscles expressing both org and tup reporter genes in late embryos attaches to the proventriculus. The PCs at the origin of this extra pair of muscles, which were named TARM*, remain to be identified (Boukhatmi, 2014).
In summary, TARMs and AMs are generated by homologous lineages and form thin, elongated muscles of characteristic morphology and attachment sites that integrate segment-specific information (Boukhatmi, 2014).
Descriptions of AMs in adults of different insects led to proposals of their role in either maintaining the position of the heart, regulating the hemolymph flow and/or controlling heart beat. The requirement of transient contacts between some AMs and the distal tip cell of MTs revealed yet another role of AMs, namely for proper bending and positioning of MTs. Through linking the lateral epidermis to the dorsal vessel, AMs press the dorsal, main branch of the trachea towards the body wall. TARMs also establish intimate topological relations with cephalic branches of the trachea in their trajectory from thoracic lateral epidermis/exoskeleton to specific regions of the midgut. Rotation of the gut at the end of embryogenesis results in stretching and bilateral asymmetry of the left and right TARMs. This suggests that TARMs have elastic properties, as already indicated by their transient deformation upon interaction with the MT tip cell (Boukhatmi, 2014).
Although the function of AMs and TARMs during larval development and growth remains to be fully assessed, the morphological and elastic properties of AMs and TARMs suggest that they could be involved in controlling the position of the heart, trachea and gut during larval foraging movements. Whereas Org-1 and Tup are only co-expressed in the AM/TARM lineage, each is expressed in several other mesodermal or non-mesodermal tissues, precluding the use of mutants to selectively study AM and TARM function. Whether AMs and TARMs represent a new type of muscle with both spring-like and contractile properties is one of the questions to be addressed henceforth (Boukhatmi, 2014).
The morphological properties specific to each Drosophila body wall muscle are determined by the expression of specific combinations of iTFs in each FC and derived muscle. Lineage specificity involves positive and negative cross-regulations between different iTFs, as well as iTF autoregulation. The maintenance of Org-1 and Tup expression in body wall muscles depends upon autoregulation (Boukhatmi, 2012; Schaub, 2012). This study shows that tup autoregulation in dorsal muscles is direct, but does not operate in AMs; here, tup is directly regulated by Org-1. Org-1 directly regulates the expression of two other iTFs, namely slouch and ladybird, in other muscle lineages (Schaub, 2012). The AM/TARM Org-1>Tup hierarchy further underlines the intricate, combinatorial nature of transcriptional regulatory networks specifying Drosophila muscle identity. This study has now identified an Org-1-dependent tup enhancer called tupAME, which is only active in AMs/TARMs and ectodermal cells connecting the frontal sac and pharynx. This CRM should provide a means to specifically target expression and modify AMs/TARMs in order to assess their function in larvae (Boukhatmi, 2014).
Nkx2.5, Tbx1 and Islet1 are major actors in the vertebrate genetic program controlling early heart and pharyngeal muscle development from common progenitors lying in the second heart field (SHF) in chordates. NK4/Nkx2.5 was recently shown to antagonize Tbx1 and repress EBF/COE function to promote cardiac versus pharyngeal muscle fate in the ascidian SHF, with Islet1 being expressed in both derivatives. In the Drosophila embryo, the orthologs of Nkx2.5 and Islet1 are required for the formation of all (in the case of Tin) or some (in the case of Tup) of the dorsal mesodermal derivatives (heart, dorsal body wall muscles, visceral muscles, lymph gland). Moreover, Tup represses the COE ortholog Collier (Col; Knot - FlyBase) in dorsal muscles, and Org-1/Tbx1 is expressed in some body wall muscles, the AMs and the visceral mesoderm, although not in myocardial progenitors. Together, these findings suggest that the Nkx2.5/Tin, Tbx1/Org-1, Islet1/Tup and COE/Col transcription factors are part of an ancestral regulatory kernel controlling diversification of heart and muscle lineages from a common progenitor pool. Future studies will be required to establish which ancestral interactions have been redirected to foster the emergence of insect AMs and TARMs (Boukhatmi, 2014).
No striated muscle has been described so far to connect the (exo)skeleton to the gut, either in insects or in vertebrates. In mammals, lung and heart are separated from visceral organs by the diaphragm muscle. However, a muscular diaphragm is a defining characteristic of mammals that is not found in other vertebrates, and the ancestral origin of this recent innovation is currently unknown. Although highly speculative, it will be interesting to investigate whether the vertebrate muscle/septum and the insect AMs/TARMs could represent two specific adaptations of an ancestral demarcation between the circulatory systems, respiratory systems and visceral organs (Boukhatmi, 2014).
optomotor-blind and optomotor-blind related-1 (org-1) encode T-domain DNA binding proteins in Drosophila. Members of this family of transcription factors play widely varying roles during early development and organogenesis in both vertebrates and invertebrates. Functional specificity differs in spite of similar DNA binding preferences of all family members. Using a series of domain swap chimeras, in which different parts of Omb and Org-1 were mutually exchanged, the relevance of individual domains were investigated in vitro and in vivo. In cell culture transfection assays, Org-1 is a strong transcriptional activator, whereas Omb appears neutral. The main transcriptional activation function was identified in the C-terminal part of Org-1. Also in vivo, Omb and Org-1 showed qualitative differences when the proteins are ectopically expressed during development. Gain-of-function expression of Omb is known to counteract eye formation and results in the loss of the arista, whereas Org-1 has little effect on eye development but causes antenna-to-leg transformations and shortened legs in the corresponding gain-of-function situations. The functional properties of Omb/Org-1 chimeras in several developmental contexts is dominated by the origin of the C-terminal region, suggesting that the transcriptional activation potential can be one major determinant of developmental specificity. In late eye development, however, a strong influence of the T-domain on ommatidial differentiation is observed. The specificity of chimeric omb/org-1 transgenes, thus, depends on the cellular context in which they are expressed. This suggests that both transcriptional activation/repression properties as well as intrinsic DNA binding specificity can contribute to the functional characteristics of T-domain factors (Porsch, 2004).
org-1, a Drosophila homologue of mammalian TBX1, is most strongly expressed during embryogenesis where it appears to play a role in the patterning of the visceral mesoderm (Lee, 2003). Expression in imaginal discs is poorly detectable. No org-1 mutants are known. Ubiquitous induction of org-1 RNA interference causes pupal lethality. Strong RNAi expression during imaginal development revealed an org-1 requirement predominantly in thorax and distal wing development but no apparent involvement in eye-antennal or leg discs. RNAi constructs from two different parts of the org-1 cDNA yielded the same phenotype indicating specificity of the interference experiment (Porsch, 2004).
The T-domain was originally defined by a statistical analysis of the Omb protein sequence and by homology to the mouse Brachyury protein. Further work has revealed the Brachyury T-domain as a sequence specific DNA binding domain, C-terminally flanked by a set of transactivation and repression domains. In vitro target site selection analyses indicate preferential Brachyury binding to a 20 bp degenerate palindrome made up of two closely related half sites. Numerous in vitro selection and in vivo transcription experiments, and the characterization of actual target genes identified the Brachyury consensus half site as a common target of all T-domain proteins investigated, so far. This also holds for Omb and Org-1. Within the enhancers of T-domain protein-controlled genes, half sites generally occur in small groups of two or more members. The half sites are variably spaced, occur in all conceivable relative orientations, and can show considerable deviation from the consensus sequence (Porsch, 2004 and references therein).
The majority of T-box factors investigated to date, are transcriptional activators. ORG-1 also functions as an activator. The only known transcriptional repressors in the T-box gene family are TBX2 and TBX3/ET, the closest vertebrate homologs of Omb. OMB, too, is a member of this functional subgroup. Furthermore, T-domain proteins can physically interact with other DNA bound factors to control target gene expression. On natural promoters, closely related T-domain proteins can replace one another while more distantly related family members are inactive or exert different effects. However, even in the same cellular context, T-domain proteins of the same subfamily can differ in developmental specificity. An example for this is provided by the role of Tbx4 and Tbx5 in limb development. Tbx4 and Tbx5 code for closely related T-domain proteins. In higher vertebrates, they are expressed in nearly complementary patterns during limb formation: Tbx5 is exclusively expressed throughout the forelimb bud, whereas Tbx4 mRNA is predominantly found in the hindlimb bud. In the chick, misexpression of Tbx5 in the presumptive hindlimb region causes a partial transformation of the leg into wing, resulting in wing/leg mosaic limbs. Conversely, ectopic Tbx4 in the developing wing promotes the growth of leg-like structures. The mechanisms underlying T-domain protein functional specificity are therefore far from understood (Porsch, 2004 and references therein).
This study addresses the question about the relative role of different parts of T-domain proteins (T-domain versus flanking regions) for their biological activity. Two distantly related Drosophila T-domain proteins (Omb and Org-1 share 61% of residues in the T-domain but are highly diverged outside) cause distinct phenotypes when ectopically expressed during fly development. This provides the basis for a domain swap experiment. Chimeras were created in which the T-domain or the T-flanking regions were exchanged between the two proteins and whether a given phenotypic effect was associated with a particular protein domain was determined. The chimeric genes were expressed in cultured cells and in transgenic flies. In transfected cells, Omb and Org-1 show a clear-cut difference in transcriptional activation potential which can be attributed largely to the origin of the C-terminal region. Also in vivo, in several tissues, the developmental consequences of chimeric gene expression were dominated by the origin of the C-terminal domain. In one developmental context (retinal differentiation), however, the developmental outcome was affected by the origin of all parts of the chimeric protein. The results demonstrate the importance of protein sequences lying both within and outside of the T-domain for the functional specificity of these T-domain proteins and show that distinct parts of Omb and Org-1 are required for their specific effects in different cellular contexts (Porsch, 2004).
Expression of omb or org-1 during imaginal disc development can interfere with normal imaginal development or promote the development of novel adult features. The phenotypic consequences of ectopic expression differed profoundly for Omb and Org-1. This observation provided the basis for an in vivo assay to investigate the significance of the T-domain versus the N- and C-terminally flanking regions in Omb and Org-1 for developmental specificity (Porsch, 2004).
The data obtained with Omb/Org-1 chimeras indicate that regions outside of the T-domain can govern chimera developmental specificity. In dpp-Gal4 driven expression, the C-terminal domains of Omb or Org-1 are sufficient to endow Omb/Org-1 chimeras with Omb-like or Org-1-like character in the early development of both eye and antenna. These data cannot be interpreted as signifying that differences in DNA binding specificity are irrelevant for the different developmental roles of the two T-domain proteins. They do suggest, however, that the intrinsic (i.e., T-domain autonomous) DNA binding specificity is not the decisive factor. Rather, the C-domains of the two proteins could differ in activation/repression potential or could specify DNA binding extrinsically by interaction with cofactors. In cell culture assays it has been shown that Omb and Org-1 drastically differ in activation potential, Org-1 being a strong activator while Omb could not activate transcription from the hsp70 minimal promoter. Due to the low basal transcription rate of the reporter gene, these experiments could not provide direct evidence for a repressive role of Omb. Two observations suggest, however, that in particular the Omb C-domain is able to act as a repressor. (1) The Omb C-terminal region contains several runs of homopolymeric alanine. This has been noted as a characteristic feature in several transcriptional repressors. (2) The low transcriptional activation by the Org-1 N-domain in the presence of the Omb C-domain and the apparent activational synergism between the Org-1 N- and C-domains suggest repressive qualities of the Omb C-domain (Porsch, 2004).
This difference in transcription activation function correlates with the dominant character of the C-domains in the Omb/Org-1 chimeras. This suggests that, in early eye-antenna development, the effects of Omb and Org-1 are governed by their distinct transcriptional activation potentials. These results do not rule out a more specific role of the C-terminal domains. This question can now be addressed using heterologous activation/repression domains. In early eye development, the phenotypic consequences of ectopic omb and org-1 expression appear as antagonistic, omb leading to a reduction, org-1 to an increase in ommatidial number. One explanation for this antagonism is that, in this tissue, Omb and Org-1 can bind to the same set of target genes, whose activation leads to an increase in ommatidial number, while their repression causes the opposite effect. There is precedence for the antagonistic action of T-domain proteins in developmental decisions. Tbx5 and Tbx2 bind to the ANF gene promoter in mammalian heart development either activating or repressing its transcription. In zebrafish mesoderm development, the T-domain proteins Ntl and Tbx6 compete for common target sites. Comparable to Org-1 and Omb, NTL is a transcriptional activator and Tbx6 has no activation potential. In a transphylum domain-swap experiment between Brachyury homologs, a quantitative determinant of mesoderm induction could also be localized to the C-terminus. In this case, it was not determined whether there was a correlation with the transactivation potential. In the same experiments, a determinant for endoderm specification was identified in the N-terminus. The relevance of the T-domain flanking domains is also apparent from the analysis of TBX5 mutations in Holt-Oram syndrome (HOS) patients (Fan, 2003). HOS can be elicited by mutations in the flanking domains, which do not affect DNA binding in vitro but completely abolish synergistic interaction with Nkx2-5 (Porsch, 2004 and references therein).
All chimeras containing the Org-1 T-domain cause an unusual hairy eye phenotype when expressed under GMR-Gal4 control indicating an involvement of T-domain DNA binding specificity. GMR-Gal4 drives gene expression in all cells of the eye disc epithelium posterior to the morphogenetic furrow. In this cell population, omb is expressed and required only at the dorso-ventral margins while org-1 appears to play no role in normal development and has not been detected. GMR-Gal4, therefore, is active in a tissue where neither gene is normally expressed. It is difficult to conceive of retinal degeneration and loss of interommatidial bristles as the two alternatives of one developmental decision effected by the antagonistic regulation of one set of target genes. In this case, it appears more likely that Omb and Org-1 ectopically bind and regulate different target genes (Porsch, 2004).
In a previous study on T-box specificity, Smith and colleagues (Conlon, 2001) investigated the relevance of the T-domains of Xbra, VegT, and Eomesodermin for determination of target gene specificity by expressing T-domain fusion proteins in early Xenopus embryos. In this case, the specificity of the three investigated proteins was determined to a large extent, but not exclusively, by the T-domain. As outlined in the introduction, all T-domain proteins are able to interact with (groups of) half sites as originally defined by Brachyury binding studies. Individual T-domain proteins can, however, differ in other binding characteristics such as dimerization tendency or the preference for certain arrangements of binding sites with regard to spacing or orientation. The replacement of a single presumably DNA binding amino acid of VegT and Eomesodermin with the corresponding amino acid of Xbra is sufficient to change the target gene expression profile of VegT and Eomesodermin to resemble that of Xbra (Conlon, 2001). This suggests that differences in DNA binding can be crucial for target gene specificity of T-domain proteins in vivo. As stated above, intrinsic DNA binding specificity as exclusive determinant of developmental specificity is not compatible with the overriding influence of the C-terminal domain which was found in some experiments (Porsch, 2004).
The phenotype of a given Omb/Org-1 chimera could be Omb-like or Org-1-like depending on the Gal4 driver under whose control it was expressed. The clearest example for this observation was provided by chimera org-1N+ombT+ombC, which, when expressed under dpp-Gal4 control, unambiguously showed omb-specific phenotypes in eye and antenna, but caused an org-1-like character in ommatidia when activated by GMR-Gal4. It is concluded from these findings that different protein domains contribute to Omb or Org-1 function in the undifferentiated eye/antennal disc versus differentiating retina cells. The C-domains of Omb and Org-1 are the main specificity determinants in Omb/Org-1 chimeras in early eye/antennal development, whereas generally all three Omb domains contribute to Omb specificity during ommatidial differentiation. This suggests that the specificity of Omb/Org-1 chimeras is not solely intrinsic to their protein sequences, but also depends on the cellular context in which they are expressed. In T-domain proteins, a cell-type dependence was described for the nucleo-cytoplasmic distribution (Collavoli, 2003) and for activation/repression properties (Stennard, 2003). The situation with T-domain proteins is thus comparable to that observed with homeodomain DNA binding proteins. In various proteins and cellular contexts a domineering influence of both the homeodomain and flanking protein domains on developmental specificity has been described. The different phenotypes of Omb/Org-1 chimeras in different cell types and developmental stages will aid in the identification of tissue- and stage-specific cofactors (Porsch, 2004 and references therein).
It was noted that certain Omb/Org-1 domain compositions give rise to novel phenotypes that are not observed with the parental proteins Omb or Org-1. For example, misexpression of org-1N+ombT+org-1C or ombN+org-1T+ombC induces caudal expansions of the eye. In the antenna, third antennal segments showed bulbous outgrowths upon expression of various chimeras. Certain omb/org-1 chimeras induced abundant ectopic microchaetae in the eye field when misexpressed with GMR-Gal4. The loss of interommatidial bristles, as caused by org-1 overexpression, is a rather common phenotype which can, for example, be observed upon changes in the signaling activity of the Notch and wingless pathways. The ectopic formation of microchaetae on the facet eye has not been described previously, in Drosophila melanogaster. However, in Drosophila robusta, the isolation of a spontaneous hairy eyed mutant has been reported which may have been phenotypically similar (Porsch, 2004).
Possible explanations for the potency of certain chimeras to produce novel phenotypes include an altered regulation of Omb or Org-1 target genes and/or new target gene specificities due to inappropriate protein-protein interactions. In the metazoan T-box proteins, the N- and C-terminal domains flanking the T-domain generally are poorly conserved, even between closely related species. This lack of constraint may explain the great versatility of T-box genes which evolved to control a wealth of biological processes (Porsch, 2004 and references therein).
Search PubMed for articles about Drosophila Org-1
Boukhatmi, H., Frendo, J. L., Enriquez, J., Crozatier, M., Dubois, L. and Vincent, A. (2012). Tup/Islet1 integrates time and position to specify muscle identity in Drosophila. Development 139: 3572-3582. PubMed ID: 22949613
Boukhatmi, H., Schaub, C., Bataille, L., Reim, I., Frendo, J. L., Frasch, M., Vincent, A. (2014). An Org-1-Tup transcriptional cascade reveals different types of alary muscles connecting internal organs in Drosophila. Development [Epub ahead of print]. PubMed ID: 25209244
Collavoli, A., Hatcher, C. J., He, J., Okin, D., Deo, R. and Basson, C. T. (2003). TBX5 nuclear localization is mediated by dual cooperative intramolecular signals. J Mol Cell Cardiol 35: 1191-1195. PubMed ID: 14519429
Conlon, F. L., et al. (2001). Determinants of T box protein specificity. Development 128: 3749-3758. PubMed ID: 11585801
Lee, H. H., Norris, A., Weiss, J. B. and Frasch, M. (2003). Jelly belly protein activates the receptor tyrosine kinase Alk to specify visceral muscle pioneers. Nature 425: 507-512. PubMed ID: 14523446
Porsch, M., Hofmeyer, K., Bausenwein, B. S., Grimm, S., Weber, B. H., Miassod, R. and Pflugfelder, G. O. (1998). Isolation of a Drosophila T-box gene closely related to human TBX1. Gene 212: 237-248. PubMed ID: 9611267
Porsch, M., Sauer, M., Schulze, S., Bahlo, A., Roth, M. and Pflugfelder, G. O. (2004). The relative role of the T-domain and flanking sequences for developmental control and transcriptional regulation in protein chimeras of Drosophila OMB and ORG-1. Mech. Dev. 122: 81-96. PubMed ID: 15582779
Schaub, C., Nagaso, H., Jin, H. and Frasch, M. (2012). Org-1, the Drosophila ortholog of Tbx1, is a direct activator of known identity genes during muscle specification. Development 139: 1001-1012. PubMed ID: 22318630
Schaub, C. and Frasch, M. (2013). Org-1 is required for the diversification of circular visceral muscle founder cells and normal midgutmorphogenesis. Dev Biol. 376(2): 245-59. Org-1 is required for the diversification. PubMed ID: 23380635
Stennard, F. A., Costa, M. W., Elliott, D. A., Rankin, S., Haast, S. J., Lai, D., McDonald, L. P., Niederreither, K., Dolle, P., Bruneau, B. G., Zorn, A. M. and Harvey, R. P. (2003). Cardiac T-box factor Tbx20 directly interacts with Nkx2-5, GATA4, and GATA5 in regulation of gene expression in the developing heart. Dev Biol 262: 206-224. PubMed ID: 14550786
date revised: 10 October 2014
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