Transcriptional Regulation

opa is positively regulated by Antennapedia and abdominal-A at the location of the first and third visceral mesodermal midgut constrictions respectively. Between these domains opa is negatively regulated by Ultrabithorax and decapentaplegic (Cimbora, 1995).

Hox genes have large expression domains, yet these genes control the formation of fine pattern elements at specific locations. The mechanism underlying subdivision of the abdominal-A (abdA) Hox domain in the visceral mesoderm has been examined. AbdA directs formation of an embryonic midgut constriction at a precise location within the broad and uniform abdA expression domain. The constriction divides the abdA domain of the midgut into two chambers, the anterior one producing the Pointed (Pnt) ETS transcription factors and the posterior one the Odd-paired (Opa) zinc finger protein. Transcription of both pnt and opa is activated by abdA. Near the anterior limit of the abdA domain, two signals, Decapentaplegic and Wingless, are produced, in adjacent non-overlapping patterns, under Hox control in mesoderm cells. AbdA is proposed to activate three targets, in distinct subsets of its broad domain of expression: wg at the anterior boundary of Connectin (Con) patch 7; pnt from anterior Con patch 7 to anterior Con patch 8, and opa, from anterior Con patch 8 through Con patch 11. Dpp signaling plays a central role in setting these distinct expression domains. The initial activation of wg by AbdA requires dpp. opa is activated in all abdA-expressing cells that do not receive a Dpp signal, defining the site of the posterior constriction. wg, in collaboration with abdA, activates pnt to generate the appropriate number of cells in the third midgut chamber, positioning the posterior constriction at the proper distance from the central constriction and partitioning the posterior midgut appropriately. Fine patterning of the posterior midgut is achieved by the activity of diffusible signals emanating from the central midgut, a remarkably long-range organizing effect (Bilder, 1998).

Hox proteins play fundamental roles in generating pattern diversity during development and evolution, acting in broad domains but controlling localized cell diversification and pattern. Much remains to be learned about how Hox selector proteins generate cell-type diversity. In this study, regulatory specificity was investigated by dissecting the genetic and molecular requirements that allow the Hox protein Abdominal A to activate wingless in only a few cells of its broad expression domain in the Drosophila visceral mesoderm. The Dpp/Tgfß signal controls Abdominal A function, and Hox protein and signal-activated regulators converge on a wingless enhancer. The signal, acting through Mad and Creb, provides spatial information that subdivides the domain of Abdominal A function through direct combinatorial action, conferring specificity and diversity upon Abdominal A activity (Grienenberger, 2003).

This study provides a conceptual framework for understanding the molecular basis of regional Hox protein transcriptional activity. Dpp and Wg signaling subdivide the AbdA Hox domain, allowing activation of pointed (pnt) and opa target genes in the third and fourth midgut chambers, respectively. Based upon the data presented here, it is suspected that the localized activation of pnt and opa by AbdA also relies on direct enhancer integration of Hox and signaling inputs. Accordingly, a Hox/signaling combinatorial code functionally subdivides the domain where a single Hox protein is made, giving rise to discrete patterns of target gene activation. The structures of relevant cis-regulatory regions of AbdA target genes are instrumental for determining which signal is required to allow activation by AbdA. The pnt midgut enhancer would contain AbdA and Wg response elements and would be activated by AbdA specifically in the third midgut chamber through the combinatorial action of AbdA and the Drosophila Tcf/Arm transcriptional effector of Wg signaling. Similarly, the opa midgut enhancer would contain AbdA and Dpp response elements and would be activated only in the fourth gut chamber by AbdA, in this case because of an inhibitory effect of the Dpp-regulated transcription factor on AbdA activity (Grienenberger, 2003).

Org-1 is required for the diversification of circular visceral muscle founder cells and normal midgut morphogenesis

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).

Targets of Activity

There are several distinct phases of runt expression in the early embryo. Each phase depends on a different set of regulators. In a third distinct phase of expression, at the onset of gastrulation, runt becomes expressed in 14 stripes. fushi tarazu plays a negative regulatory role in generating this pattern, whereas the pair-rule genes paired and odd-paired are required for activating or maintaining runt expression during these stages (Klingler, 1993).

The odd-paired gene is essential for parasegmental subdivision of the Drosophila embryo. opa is required for the activation of wingless and engrailed in all parasegments. OPA does not act in a spatially restricted manner to establish the position of en and wg expression. Because of its ubiquitous expression, OPA must cooperate with other spatially restricted proteins to achieve proper pair-rule subdivision of the Drosophila embryo (Benedyk, 1994).

The exact positioning of neuroblasts in the neuroectodermal region that gives rise to the CNS is regulated by a combination of pair-rule genes. Proneural achaete-scute genes are controlled by combinations of axis-patterning genes through a common intergenic control region. Specifically, in every segment, the loss of odd-paired function removes achaete expression from the second row of clusters in each segment (Skeath, 1992).

opa regulates bagpipe in the visceral mesoderm (Cimbora, 1995).

Acting either via opa or in concert with it, Tenascin major, the extracellular protein related to vertebrate tenascin, initiates a signal transduction cascade which acts on downstream targets such as paired, sloppy-paired, gooseberry, engrailed and wingless, leading to an opa-like phenotype (Baumgartner, 1994).

DPTP61F is a non-receptor protein tyrosine phosphatase that is expressed during Drosophila oogenesis and embryogenesis. DPTP61F transcripts are alternatively spliced to produce two isoforms of the protein which are targeted to different subcellular locations. The transcript encoding DPTP61Fm accumulates in 16 segmentally repeated stripes in the ectoderm during germband extension. These stripes are flanked by, and adjacent to, the domains of engrailed and wingless gene expression along the anterior/posterior axis. In stage 10 embryos, the domains of DPTP61Fm transcript accumulation are wedge shaped and roughly coincide with the area lateral to the denticle belts that will give rise to naked cuticle. The DPTP61Fm transcript is also expressed later in embryogenesis in the central nervous system. The segmental modulation of DPTP61Fm transcript accumulation along the A/P axis of the germband is regulated by the pair-rule genes, and the intrasegmental pattern of transcript accumulation is regulated by the segment polarity genes. In hairy mutants, the complement of DPTP62Fm stripes is reduced by half, to approximately eight wide stripes. It is presumed that odd numbered stripes have been deleted. Within embryos homozygous for a strong eve allele, odd stripes are absent except for stripe 1. In odd paired mutants every even stripe is decreased. In paired mutants odd numbered domains of expression are shifted anteriorly towards the even numbered domains. wingless, hedgehog, naked and patched are involved in refining the pattern of mRNA accumulation within each parasegment (Ursuliak, 1997).

cis-regulatory requirements for tissue-specific programs of the circadian clock

Broadly expressed transcriptions factors (TFs) control tissue-specific programs of gene expression through interactions with local TF networks. A prime example is the circadian clock: although the conserved TFs Clock (Clk) and Cycle (Cyc) control a transcriptional circuit throughout animal bodies, rhythms in behavior and physiology are generated tissue specifically. Yet, how Clk and Cyc determine tissue-specific clock programs has remained unclear. This study used a functional genomics approach to determine the cis-regulatory requirements for clock specificity. First Clk and Cyc genome-wide binding targets in heads and bodies were determined by ChIP-seq, and they were shown to have distinct DNA targets in the two tissue contexts. Computational dissection of Clk/Cyc context-specific binding sites reveals sequence motifs for putative partner factors, which are predictive for individual binding sites. Among them, it was shown that the opa and GATA motifs, differentially enriched in head and body binding sites respectively, can be bound by Opa and Serpent (Srp). They act synergistically with Clk/Cyc in the Drosophila feedback loop, suggesting that they help to determine their direct targets and therefore orchestrate tissue-specific clock outputs. In addition, using in vivo transgenic assays, it was validated that GATA motifs are required for proper tissue-specific gene expression in the adult fat body, midgut, and Malpighian tubules, revealing a cis-regulatory signature for enhancers of the peripheral circadian clock. These results reveal how universal clock circuits can regulate tissue-specific rhythms and, more generally, provide insights into the mechanism by which universal TFs can be modulated to drive tissue-specific programs of gene expression (Meireles-Filho, 2013).

Although frequently not restricted to single cell types, individual TFs can control tissue-specific programs of gene expression through interactions with local TF networks. But despite substantial progress in identifying differential cell-specific circadian expression programs, how Clk and Cyc interact with local TF networks to generate output rhythms tissue specifically is still elusive (Meireles-Filho, 2013).

This study used an integrative genomics approach to shed light on how the circadian clock drives tissue-specific gene expression. While shared Clk/Cyc binding sites could not be explained by combinations of head- and body-specific motifs, yet were slightly more enriched in E box motifs and -- similar to highly occupied target [HOT] regions -- in Trithorax-like motifs [Trl/GAGA; 2-fold]), a substantial number of Clk and Cyc binding sites were specific to either heads or bodies and next to genes with different functional GO categories. These binding sites differed substantially in their motif content, and this motif signature was predictive of context-specific Clk/Cyc binding, suggesting that tissue-specific clock targets are determined by the binding site sequences (Meireles-Filho, 2013).

GATA motifs were enriched in Clk/Cyc binding sites in bodies and required for enhancer activity in the fat body, midgut, and Malpighian tubules. This suggests that GATA factors might play a key role for Clk/Cyc-bound enhancers in bodies, potentially by helping to establish the chromatin landscape in tissues where they are specifically expressed (e.g., srp in the fat body and GATAe in the gut). Interestingly, GATA motifs are also overrepresented in promoter regions of circadian genes in rodents, suggesting a conserved role for GATA factors in the circadian clock (Meireles-Filho, 2013).

This study found that the GATA factor Srp could act synergistically with Clk, suggesting that it is an important determinant of clock function in peripheral tissues. Srp has multiple functions in Drosophila, including the control of endodermal development and hematopoiesis in the embryo and the induction of immune response in the larval fat body. Interestingly, srp is coexpressed with Clk and Cyc in the fat body, a tissue with roles in metabolic activity, innate immunity response, and detoxification - all known to be controlled in a circadian manner. Clk body-specific peaks were 4.17-fold enriched close to cycling fat body genes, suggesting that srp might help determine the physiological outputs controlled by the fat body pacemaker. Interestingly, srp is also required for hormone-induced expression of the Fbp1 TF during fat body development, supporting the idea that it might be important for temporal or inducible regulation more generally (Meireles-Filho, 2013).

Similarly, Opa, which belongs to the Zic family of mammalian TFs with conserved roles in head formation in flies and mammals, is coexpressed with Clk and cyc in the adult brain. In addition, an enhancer of Slob, an output gene of the clock pacemaker involved in the generation of locomotor activity rhythms, responded to Clk and Cyc in an Opa-dependent manner, suggesting that Opa might be involved in the recruitment of Clk/Cyc to regulate genes controlling fly behavior. Further studies on Opa and additional predicted partner TFs might provide new insights into the Drosophila clock in the head (Meireles-Filho, 2013).

It is likely that different cofactors with functions equivalent to srp or opa exist in different cell types, which redirect Clk/ Cyc to tissue-specific binding sites and allow tissue-specific gene regulation. Indeed, this study has identified several other motifs that are tissue-specifically enriched. This is reminiscent of studies showing that TFs downstream of signaling pathways are redirected in a tissue-specific manner by cell-specific master regulators. The results might thus constitute an important example of how partner TFs adapt broadly active transcriptional regulators to achieve tissue-specific gene expression and function, contributing to a better understanding of gene regulatory networks more generally (Meireles-Filho, 2013).

These data on Clk/Cyc binding in different contexts not only provide novel insights into clock regulatory networks and enhancer structure but also exemplify a new strategy to uncover cofactors of the circadian clock via their cis-regulatory motifs. This approach is complementary to forward and reverse genetics or biochemistry, which have traditionally been used to reveal clock factors. It can also be applied more generally to identify factors that recruit broadly expressed TFs in different cell types or tissues. In addition, the tagging of endogenous loci allows the study of TFs under physiological conditions in their endogenous expression domains, which is crucial especially for TFs that have large and complex regulatory regions and/or for which physiological expression levels are of fundamental importance. In summary, the results in the Drosophila circadian clock reveal how universal TF circuits can be modulated to generate transcriptional tissue-specific outputs and demonstrate a novel approach to determine regulatory partners more generally (Meireles-Filho, 2013).

odd-paired: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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