InteractiveFly: GeneBrief

Dorsocross1, Dorsocross2 and Dorsocross3: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Signals from the BMP family member Decapentaplegic (Dpp) play a role in establishing a variety of positional cell identities in dorsal and lateral areas of the early Drosophila embryo, including the extra-embryonic amnioserosa as well as different ectodermal and mesodermal cell types. Although a reasonably clear picture is available of how Dpp signaling activity is modulated spatially and temporally during these processes, a better understanding of how these signals are executed requires the identification and characterization of a collection of downstream genes that uniquely respond to these signals. Three novel genes, Dorsocross1, Dorsocross2 and Dorsocross3, are described that are expressed downstream of Dpp in the presumptive and definitive amnioserosa, dorsal ectoderm and dorsal mesoderm. These genes are good candidates for being direct targets of the Dpp signaling cascade. Dorsocross expression in the dorsal ectoderm and mesoderm is metameric and requires a combination of Dpp and Wingless signals. In addition, a transverse stripe of expression in dorsoanterior areas of early embryos is independent of Dpp. The Dorsocross genes encode closely related proteins of the T-box domain family of transcription factors. All three genes are arranged in a gene cluster, are expressed in identical patterns in embryos, and appear to be genetically redundant. By generating mutants with a loss of all three Dorsocross genes, it has been demonstrated that Dorsocross gene activity is crucial for the completion of differentiation, cell proliferation arrest, and survival of amnioserosa cells. In addition, the Dorsocross genes are required for normal patterning of the dorsolateral ectoderm and, in particular, the repression of wingless and the ladybird homeobox genes within this area of the germ band. These findings extend knowledge of the regulatory pathways during amnioserosa development and the patterning of the dorsolateral embryonic germ band in response to Dpp signals (Reim, 2003).

Cell identities that are determined by Dpp include the dorsal epidermis and peripheral nervous system (PNS) in dorsolateral regions of the ectoderm, the dorsal vessel, dorsal somatic and visceral muscles in the dorsal mesoderm as well as those of the extra-embryonic amnioserosa in the dorsalmost region of the embryo. In addition to promoting dorsal epidermal and PNS fates in the dorsolateral ectoderm, Dpp acts to suppress the formation of neurons of the central nervous system in the same area (Reim, 2003).

For a better understanding of these activities, it is important to consider that Dpp exercises some of its functions sequentially at different stages of development, during which dpp changes its own pattern of expression. In particular, during blastoderm and gastrulation stages, Dpp acts in a dose-dependent fashion to establish positional information in dorsal and lateral areas of the embryo and to specify amnioserosa tissue. Although dpp is expressed uniformly around ~40% of the dorsal circumference of the embryos during this stage, the activity of Dpp is modulated along the dorsoventral axis by diffusion of secreted gene products as well as by positive and negative regulators of the signaling pathway. Negative regulators include Short gastrulation (Sog) and Brinker (Brk), both of which are expressed ventrolaterally. Whereas Sog and its vertebrate homolog Chordin are secreted molecules that inhibit BMP signaling via binding to the ligand, Brk appears to be a nuclear factor that interferes with the signaling output via binding to regulatory sequences of Dpp target genes. By contrast, specification of amnioserosa fates in the dorsal 10% of embryonic cells requires maximal signaling activities that involve Sog as a positive regulator of Dpp in conjunction with Twisted gastrulation (Tsg) as well as a second, uniformly-distributed BMP ligand, Screw (Scw). Dpp, Sog and Tsg are thought to be present in a diffusible trimolecular complex that serves to carry and release active Dpp prefentially into dorsalmost areas where tsg is expressed (Reim, 2003).

After gastrulation, dpp expression ceases in the developing amnioserosa and becomes restricted to a broad stripe of cells in the dorsolateral ectoderm along the elongated germ band. During this period, the dorsally migrating cells of the mesoderm reach the dpp-expressing area of the ectoderm, thus allowing Dpp to induce dorsal mesodermal cell fates across germ layers. In addition, Dpp is thought to act in the continuing patterning processes within the dorsolateral ectoderm during this stage that lead to the specification of tracheal as well as particular epidermal and sensory organ progenitors. Both in the dorsal mesoderm and dorsolateral ectoderm, Dpp must act in combination with additional patterning molecules that provoke differential responses of cells to the Dpp signal. For example, in the dorsal mesoderm, the presence or absence of Wingless (Wg) activity determines whether cells will respond to Dpp by forming heart and dorsal somatic muscle progenitors versus visceral muscle progenitors (Reim, 2003).

In order to obtain more insight into the mechanisms of how Dpp signals pattern the embryo and how they are integrated with other patterning processes, it is crucial to study the regulation of Dpp target genes. To date, detailed molecular studies have been described for three targets that are induced during early embryogenesis, namely the homeobox genes zerknüllt (zen), tinman (tin) and even-skipped (eve). zen is required for the specification of the amnioserosa downstream of Dpp. Accordingly, the expression of zen in a dorsal on/ventral off pattern, although initially Dpp-independent, requires low levels of Dpp activity for its maintenance and high Dpp activities for its subsequent refinement to areas of the prospective amnioserosa. Likewise, tin is required for the specification of all dorsal mesodermal tissues and eve for the normal differentiation of specific pericardial cells and dorsal somatic muscles in a Dpp-dependent manner. All three genes have in common the presence of multiple binding sites for intracellular Dpp effectors, the Smad proteins Mad and Medea, in their regulatory regions: the Smad proteins are essential for mediating the inductive activity of Dpp. However, in addition to these Smad-binding sites, each of these genes has a characteristic set of additional regulatory sequences that, at least in part, explains its particular spatial and tissue-specific response to Dpp signals. For example, zen contains binding sites for Brk in addition to the Smad sites. It appears that the antagonistic activities of the Brk and Smad sites and the differential ratios of Brk versus active Smad proteins along the dorsoventral embryo axis determine the ventral border of Dpp-dependent zen domain during cellularization stages. The Smad sites but not the Brk sites are also required for zen induction in the prospective amnioserosa during the cellularized blastoderm stage. The mesodermal Dpp targets tin and eve require Smad-binding sites and, in addition, binding sites for Tin, which serve to target the Dpp response to the mesoderm. Further, the Dpp-responsive enhancer of eve contains functionally important binding sites for regulators that restrict its activity to segmental subsets of dorsal mesodermal cells, including the Wg effector Pangolin (Reim, 2003).

Three novel genes have been discovered that respond to Dpp signals in the prospective amnioserosa, dorsal ectoderm and dorsal mesoderm, and are good candidates for being direct targets of the Dpp signaling cascade. The three genes, Dorsocross1 (Doc1), Dorsocross2 (Doc2) and Dorsocross3 (Doc3), which are present in a gene cluster, are closely related members of the T-box family of genes and presumably arose by relatively recent duplications from a common ancestor. The Dorsocross (Doc) genes are expressed in essentially identical patterns within several areas that receive high levels of Dpp signals, including the prospective amnioserosa during the cellularized blastoderm stage, the dorsolateral ectoderm and dorsal mesoderm during germ band elongated stages and areas that span the compartment border in wing discs. Doc expression in the prospective amnioserosa depends on dpp and zen, whereas the metameric expression in the dorsolateral ectoderm and dorsal mesoderm depends on a combination of dpp and wg. Genetic analysis demonstrates that the three Doc genes have largely redundant functions during amnioserosa development, as well as during dorsolateral ectoderm and dorsal mesoderm patterning. A focus was placed on the role of the Doc genes in the amnioserosa and dorsolateral ectoderm. They are essential for full differentiation and maintenance of the amnioserosa, including the arrest of cell proliferation in this tissue. Owing to the requirement of a functional amnioserosa for normal germ band retraction, loss of Doc activity produces embryos with a permanently extended germ band. Hence, Doc genes are functionally related to U-shaped and similarly expressed genes. These genes of the 'u-shaped group' include hindsight (hnt/pebbled), serpent (srp), tail-up (tup), u-shaped (ush), epidermal growth factor receptor (Egfr) and insulin-like receptor (InR); all are components of a regulatory network that controls normal development and functioning of the amnioserosa. In addition to the amnioserosa, Doc genes are required for the normal patterning of the dorsolateral ectoderm, which includes the repression of wg and ladybird (lb) expression within this area. These findings provide valuable insight into the mechanisms of how Dpp signals are executed during the development of the amnioserosa and the patterning of dorsolateral areas of the embryonic germ band (Reim, 2003).

The closely related T-box sequences, genomic clustering and virtually identical expression patterns of the three Dorsocross genes suggest that they are derived from relatively recent duplications of a common progenitor gene. Accordingly, the observation that loss of Doc1 or Doc3 does not cause any of the embryonic phenotypes seen upon loss of all three genes indicates that there is a large degree of functional redundancy among these three genes. Phylogenetic analysis with the extended T-box domain sequences shows that the Doc genes are most closely related to the vertebrate Tbx6 genes, whose expression in the paraxial mesoderm is reminiscent of the expression of the Doc genes in the dorsal somatic mesoderm. However, the limited reliability of the branches separating the Tbx6, VegT and Tbx2 subfamilies in the phylogenetic tree analysis, the absence of Drosophila orthologs of VegT and Tbx4/5 genes, as well as shared features of expression in the somatic and/or precardiac and cardiac mesoderm seem to support the alternative possibility that the Doc, Tbx6, VegT and Tbx4/5 genes arose from a common ancestral gene by gene amplifactions after the divergence of the insect and vertebrate lineages (Reim, 2003).

A prominent feature of the Doc genes is their expression in areas that receive inputs from Dpp, including the dorsalmost cells in blastoderm embryos, the dorsolateral ectoderm and mesoderm in the elongated germband, and distinct domains spanning the compartment border of the wing disc. Indeed, genetic data, together with the co-localization of Doc transcripts with active Mad in dorsal embryonic tissues, favor the possibility that the Doc genes are direct targets of the Dpp signaling cascade. However, the Dpp signals are required to act in combination with additional regulators during each of these inductive events (Reim, 2003).

Robust and stable induction of Doc expression in a dorsal stripe requires the activity of the homeodomain protein Zen as a co-activator of Dpp signals. The zen gene features an early, broad expression domain along the dorsal embryonic circumference, which is initially Dpp independent but subsequently requires Dpp for it to be maintained. Thereafter, its expression refines into a narrow dorsal domain in a process that requires peak levels of Dpp. The activation of Doc expression occurs at the same time as the refinement of zen expression and within the same narrow domain, which also coincides with high phospho-Mad levels. Although the maintenance and refinement of zen by Dpp is zen independent, it is proposed that Zen synergizes with peak signals of Dpp to trigger Doc gene expression in a dorsal stripe. The requirement for this proposed interaction between zen and dpp would explain the failure of zen to activate Doc genes in an early, broad domain as well as the observed low levels of residual Doc expression in zen mutant embryos, that may be due to inputs from Dpp alone. Formally, this proposed mechanism would be analogous to previously described inductive events in the early dorsal mesoderm, where the synergistic activities of the homeodomain protein Tinman and activated Smads induce the expression of downstream targets such as even-skipped. The identification of functional binding sites for Zen and Smads in Doc enhancer element(s) will be necessary for demonstrating that an analogous mechanism is active during induction of Doc gene expression in a dorsal stripe. In the absence of such data, it cannot be completely ruled out that dorsal Doc expression is controlled indirectly by Dpp, possibly via the combinatorial activities of zen and another high-level target gene of Dpp. Since mutations in several other genes that are expressed in the early amnioserosa, including pannier (pnr), hnt, srp, tup and ush, do not affect Doc expression until at least stage 12, these genes can be excluded as candidates for early upstream regulators of Doc (Reim, 2003).

Unlike zen, which is expressed only transiently, Doc expression is maintained throughout amnioserosa development. Hence, the Doc genes provide a functional link between the early patterning and specification events in dorsal areas of the blastoderm embryo and the subsequent events of amnioserosa differentiation. The activity of zen is required for all aspects of amnioserosa development that have been examined to date, including normal activation of C15. By contrast, the data demonstrate that the Doc genes execute only a subset of the functions of zen, which include the activation of Kr and hnt, but not that of C15 and early race, in amnioserosa cells. This interpretation is consistent with the failure to obtain a significant increase of amnioserosa cells upon ectopic expression of any of the Doc genes in the ectoderm or throughout the early embryo (using e22c and nanos-GAL4 drivers, respectively). The residual expression of hnt in some amnioserosa cells of Doc mutant embryos could be due to direct inputs from zen itself or from a yet undefined zen downstream gene acting in parallel with Doc. Nonetheless, the strong reduction of hnt expression in Doc mutant embryos could largely account for their amnioserosa-related phenotypes, including the absence of Kr expression, the decline of race expression, premature apoptosis and failure of germ band retraction. All of these phenotypes have also been observed in hnt mutant embryos. However, it is likely that Doc gene activity is required for the activation not only of hnt but also of additional genes of the u-shaped group and that Doc genes exert some of their functions in parallel with hnt. Some evidence for this notion is derived from the observation that loss of Doc activity has a stronger effect on Kr expression than loss of hnt activity (Reim, 2003).

One of the hallmarks of amnioserosa development is that the cells of this tissue never resume mitotic divisions after the blastoderm divisions. To a large extent, this cell cycle arrest is due to the absence of expression of cdc25/string in the prospective amnioserosa: this absence prevents the cells from entering M-phase and leads to G2 arrest. In addition, the expression of the Cdk inhibitor p21/Dacapo in the early amnioserosa is thought to contribute to the cell cycle arrest. Although a detailed description of the regulation of string and dacapo expression in dorsal embryonic areas is lacking, it has been reported that zen is required for repressing dorsal string expression -- this repression is expected to prevent further cell divisions. Notably, the observation that amnioserosa cells re-enter the cell cycle in Doc mutant embryos demonstrates that Doc genes are required for the cell cycle block in addition to zen. Whereas zen mutant embryos already feature ectopic cell divisions in dorsal areas from stage 8 onwards, in Doc mutants the amnioserosa cells resume mitosis only during and after stage 10, which is shortly after Zen protein disappears. Thus, it is hypothesized that the Doc genes take over the function of zen in repressing string and prevent cell divisions at later stages of amnioserosa development when Zen is no longer present. Overall, the phenotype of Doc mutant embryos suggests that amnioserosa differentiation, including cell cycle arrest and the development of squamous epithelial features, initiates in the absence of Doc activity but is not maintained beyond stage 11. Thereafter, cell division resumes and there is a reversal of the partially differentiated state. Apoptotic events are not observed prior to stage 11 in Doc mutants. However at later stages, many amnioserosa cells die prematurely and the remaining cells are difficult to distinguish morphologically from dorsal ectodermal cells (Reim, 2003).

Altogether, these studies have identified the Doc genes as new members of the u-shaped group of genes, which control amnioserosa development, and provide new insights into the regulatory pathways in amnioserosa development downstream of Dpp. In future studies, it will be necessary to define in more detail the specific roles of the remaining genes of the u-shaped group, particularly ush, srp, tup and C15, in this regulatory framework (Reim, 2003).

Unlike in the presumptive amnioserosa, not all cells in the dorsolateral ectoderm and dorsal mesoderm that receive high levels of Dpp induce Doc expression. Rather, Wg signals are required in combination with Dpp in these tissues, such that the Doc genes are induced at the intersections of transverse Wg stripes and the dorsally restricted domain containing high phospho-Mad levels. The Doc stripes extend beyond the peak levels of Wg on both sides of the Wg stripes, which indicates that the Doc genes are able to respond to relatively low levels of diffusible Wg. In addition, the absence of Doc expression in the dorsalmost cells of the ectoderm that receive Wg and Dpp signals indicates the presence of a negative regulator that prevents Doc induction in the ectoderm adjacent to the amnioserosa until stage 12. Together, these inputs restrict Doc expression to metameric quadrants that encompass the areas of the dorsolateral ectoderm between the tracheal placodes as well as the underlying mesodermal cells (Reim, 2003).

Some of the effects of wg are known to be mediated by its target gene sloppy paired (slp), including the feedback activation of wg in the ectoderm and the repression of bagpipe (bap) in the mesoderm. However, the residual (although strongly reduced) expression of the Doc genes in the germ band of slp mutant embryos argues against a role of slp in mediating the function of wg to induce the Doc genes. Hence, the Doc genes may be direct targets of the Wg signaling cascade in the ectoderm and mesoderm (Reim, 2003).

Observations show that one of the important functions of the Doc genes in the dorsolateral ectoderm is the repression of wg expression. Although the expression of Doc initially depends on wg, the Doc genes subsequently exert a negative feedback on wg expression, which leads to the previously unexplained interruption of the wg stripes during stage 11. Because the ventral extent of the ectodermal Doc domains correlate with the ventral borders of high levels of P-Mad, it is concluded that the dorsal limit of the ventral wg stripes at stage 11 is determined indirectly by Dpp via Doc (Reim, 2003).

The maintenance of wg after stage 10 has been shown to depend on two different positive feedback loops, one being active in the dorsal and the other in the ventral ectoderm. The dorsal feedback loop is mediated by the ladybird homeobox genes (lb=lbe and lbl), whereas the ventral loop is mediated by the Pax gene gooseberry (gsb). The Doc genes must interrupt one or both of these feedback loops, although it is not clear whether the primary block is at the level of the wg gene or at the level of the transcription factor-encoding genes lb and/or gsb. Another target for repression by the Doc genes in this pathway could be slp, which is required both dorsally and ventrally in wg feedback regulation. It is thought that lb is unlikely to be the primary target of Doc repression since the failure of wg repression temporally precedes the expansion of the lb stripes in Doc mutant embryos. Furthermore, the observation that the Doc genes can also repress wg in other tissue contexts such as the imaginal discs, where gsb, lb and slp are not components of a wg feedback loop, seems to favor the mechanism of a direct repression of the wg gene by Doc (Reim, 2003).

Taken together, these observations show that dynamic interactions among positive and negative feedback loops, which share wg as a common component, are involved in the dorsoventral and anteroposterior patterning of the embryonic ectoderm. The activity of the Doc genes in negatively regulating wg and lb, as well as their potential positive effects on yet unknown targets in the dorsolateral ectoderm, are expected to be important for the proper dorsoventral organization of the cuticle and sensory organs. In the mesoderm, the metameric expression domains of the Doc genes during stages 9-11 include the dorsal somatic and cardiac mesoderm. Notably, preliminary analysis has revealed defects in dorsal somatic muscle and dorsal vessel development in Doc mutant embryos, that are currently being examined in more detail. Finally, it is noted that the expression pattern of the Doc genes in the embryonic epidermis is very reminiscent of the pattern of expression and activity of another T-box gene, optomotor-blind (omb), in the pupal epidermis. Doc and omb expression overlap in the wing discs although, unlike omb, Doc expression is interrupted near the Wg domains. Furthermore, it has been reported that dominant mutations in the gene Scruffy (Scf) and their revertants genetically interact with omb during abdominal cuticle and wing patterning (Kopp, 1997). Because the breakpoints of two Scf revertants, Df(3L)Scf-R6 and Scf-R11, have been mapped directly upstream and downstream, respectively, of the Doc3 gene, it is tempting to speculate that the Scf phenotype is caused by rearranged Doc3. Future studies will clarify the relationship between Scf and Doc genes and establish whether the T-box genes Doc and omb functionally interact during patterning of the adult cuticle and wings (Reim, 2003).

Anterior CNS expansion driven by brain transcription factors

During CNS development, there is prominent expansion of the anterior region, the brain. In Drosophila, anterior CNS expansion emerges from three rostral features: (1) increased progenitor cell generation, (2) extended progenitor cell proliferation, (3) more proliferative daughters. This study finds that tailless (mouse Nr2E1/Tlx), otp/Rx/hbn (Otp/Arx/Rax) and Doc1/2/3 (Tbx2/3/6) are important for brain progenitor generation. These genes, and earmuff (FezF1/2), are also important for subsequent progenitor and/or daughter cell proliferation in the brain. Brain TF co-misexpression can drive brain-profile proliferation in the nerve cord, and can reprogram developing wing discs into brain neural progenitors. Brain TF expression is promoted by the PRC2 complex, acting to keep the brain free of anti-proliferative and repressive action of Hox homeotic genes. Hence, anterior expansion of the Drosophila CNS is mediated by brain TF driven 'super-generation' of progenitors, as well as 'hyper-proliferation' of progenitor and daughter cells, promoted by PRC2-mediated repression of Hox activity (Curt, 2019).

A striking feature of the central nervous system (CNS) is the significant anterior expansion of the brain relative to the nerve cord. Anterior CNS expansion is evolutionarily conserved, becoming increasingly pronounced in vertebrates, and is particularly evident in mammals with the dramatic expansion of the telencephalon. The anterior expansion of the CNS is of importance as it likely underlies the increasingly complex behaviours governed by the CNS during evolution. However, the underlying mechanisms and genetic pathways driving anterior CNS expansion are not well understood (Curt, 2019).

Drosophila melanogaster is a powerful model system for addressing this issue. The Drosophila larval CNS arises from ~ 1200 stem cells, neuroblasts (NBs), which delaminate from the neuroectoderm during early- to mid-embryogenesis, herein referred to as brain (B1-B3), subesophageal region (S1-S3) and nerve cord (T1-T3 and A1-A10) segments. The anterior-most brain segment, B1 (protocerebrum), displays much more extensive NB generation and contains more than twice the number of NBs found in any posterior segment (Curt, 2019).

In the nerve cord most NBs initiate lineage progression in the Type I mode, generating daughters that divide once to generate two neurons/glia. Subsequently, many NBs switch to the Type 0 mode, generating directly differentiating daughters. In the brain, most NBs appear to stay in the Type I mode throughout neurogenesis and furthermore proliferate for a longer time than NBs in the nerve cord. Moreover, two additional and even more proliferative modes of NB behaviour exist in the B1 region: Type II NBs and mushroom body NBs (MBNB). The Type II NBs, eight in each B1 brain lobe, bud off daughter cells, denoted intermediate neural progenitor cells (INPs), which divide multiple times, budding off daughter cells that in turn divide once to generate two neurons/glia. The MBNBs, four in each B1 brain lobe, do not appear to bud off INPs, and given the size of the MBNB lineages; around 30-40 cells by late embryogenesis it is likely that they progress in Type I mode, rather than Type 0. However, in contrast to the Type I and II NBs, and most if not all other NBs in the entire CNS, MBNBs never enter quiescence and instead continue dividing throughout embryogenesis and larvae stages. The presence of these three 'hyper-proliferative' modes of NB behaviour; the non-switching and extended proliferation Type I NBs, the Type II NBs and the never-stopping MBNBs, results in the generation of much larger average lineages in the brain at the end of embryogenesis. When combined with the 'super-generation' of NBs in B1, this conduces to the generation of far more cells in the brain segments than in more posterior ones (Curt, 2019).

The 'super-generation' of progenitors, specifically evident in the anterior-most brain segment (B1) is not well understood, but the head gap genes tailless (tll) and ocelliless [oc; also known as orthodenticle (otd)], are known to play a central role. The enhanced anterior proliferation is promoted by the action of the Polycomb Repressor Complex 2 (PRC2) epigenetic complex; the key mediator of the repressive H3K27me3 mark. PRC2 plays an essential role as a suppressor of anterior Hox homeotic gene expression. PRC2 thus ensures that the brain is free of Hox gene expression, thereby preventing the known anti-proliferative function of Hox genes. However, the role of head gap genes, and other potential brain pro-proliferative genes, with regard to the brain-specific features of proliferation are not well understood. Moreover, how brain proliferation drivers intersect with the PRC2-Hox system has not been extensively addressed (Curt, 2019).

To identify genes promoting anterior CNS expansion, a number of criteria were applied to focus in on 14 transcription factors (TFs) specifically expressed in the developing embryonic brain. From these, two genes and two gene families, denoted 'brain TFs' herein, were identified that were found to be necessary for brain NB generation and for brain-type NB and daughter cell proliferation. These include the head gap gene tll, as well as the otp/Rx/hbn, Doc1/2/3 and erm genes. Brain TF co-misexpression could drive NB and daughter cell proliferation in the nerve cord, and was sufficient to reprogram developing embryonic ectoderm and wing disc cells into brain NBs. Brain TF expression is promoted by the PRC2 complex, the main role of which is to keep the brain free of the anti-proliferative and repressive action of the Hox homeotic genes. Strikingly, the reduced brain proliferation observed in PRC2 mutants could be rescued by combinatorial expression of brain TFs (Curt, 2019).

Detailed analysis of Drosophila CNS development has revealed that there is 'super-generation' of NBs in the B1 segment; ~160 NBs in B1 compared to 28-70 NBs/segment for each of the 18 posterior segments (B2-A10). In the ventral neurogenic regions (generating the nerve cord) a single NB delaminates from each proneural cluster. In contrast, the NB super-generation in B1 stems, at least in part, from group delamination of NBs. The specification of NB cell fate depends upon low, or no, Notch activity. In line with this notion, evidence points to reduced Notch signalling in the procephalic neuroectoderm (Curt, 2019).

Head gap genes, such as tll, were previously shown to be important for B1 NB generation, and in line with this strikingly reduced NB generation in tll is observed. Does tll intersect with Notch signalling? tll mutants show loss of expression of the proneural gene l'sc, which is negatively regulated by Notch. Recent studies furthermore reveal an intimate interplay between tll and Notch signalling in the developing Drosophila embryonic optic placodes. In addition, the C. elegans tll orthologue nhr-67 regulates both lin-12 (Notch) and lag-2 (Delta) during uterus development (Verghese, 2011). Strikingly, in the mouse brain, the tll orthologue Nr2E1 (aka Tlx) was recently shown to negatively regulate the canonical Notch target gene Hes1. Against this backdrop, it is tempting to speculate that the group NB delamination normally observed in the procephalic region results, at least in part from tll repression of the Notch pathway. Indeed, tll was the only one of the four TFs that could act alone to trigger ectopic NBs in the wing disc (Curt, 2019).

Other previously identified head gap genes are oc and ems. However, misexpression of oc or ems from elav-Gal4 bid not efficiently drive ectopic proliferation in the nerve cord. Moreover, oc acts both in B1-B2, ems in B2-B3, being repressed from B1 by tll, and btd acts in B2-B3. Because B2 and B3 segments do not display super-generation of NBs these findings point to tll as the key head gap gene driving the super-generation of NBs specifically observed in the B1 segment (Curt, 2019).

Reduced NB generation was observed in the triple otp/Rx/hbn and Doc1/2/3 mutants. This would tentatively place them in the category of head gap genes, at least as far as being important for NB generation. However, their effects on NB generation is weaker than that observed in tll mutants. In addition, otp/Rx/hbn and Doc1/2/3 show genetic redundancy. The combination of genetic redundancy and their weaker effects on NB generation, likely explain why they were not previously categorised as head gap genes (Curt, 2019).

The connection between the brain TFs studied herein and NB super-generation is not only evident from the mutant phenotypes, but also from their potent gain-of-function effects. Strikingly, it was found that brain TF co-misexpression was sufficient to generate ectopic NBs in the embryonic ectoderm and developing wing discs. A number of markers indicate that these ectopic NBs undergo normal CNS NB lineage progression, generating neurons and glia. Moreover, the ectopic expression of the brain-specific factors Rx and Hbn, the apparently higher neuron/glia ratio, the reduced GsbN expression, the generation of Dpn+/Ase- NBs (Type II-like) in both the embryonic ectoderm and wing discs, in combination suggest that brain TF co-misexpression specifically triggered reprogramming towards a B1 brain-like phenotype (Curt, 2019).

One surprising finding pertains to the clear difference between the potency of the different 'double' and 'Tetra' UAS combinations of tll,erm in the embryonic ectoderm versus the wing disc, with the double being more potent in the wing disc and the Tetra more potent in the embryo. Indeed, in the wing disc the strong effect of tll,erm is suppressed by the addition of any combination of otp and Doc2. There is no obvious explanation for the different responsiveness to brain TF misexpression in the two tissues, but it may reflect the fact the embryonic neuroectoderm is already primed for the generation of NBs (Curt, 2019).

Another surprising finding pertains to the role of erm in embryonic versus larva Type II NBs. Previous studies of erm function in the larvae found that erm mutants displayed more Type II NBs. Larval MARCM clone induction and marker analysis demonstrate that this is due to de-differentiation of INPs back to type II NBs, rather than excess generation of Type II NBs in the embryo. Extra Type II or Type I NBs in were not found in erm mutants but rather reduced number of cells generated in the embryonic Type II lineages, showing that erm is important for lineage progression. Hence, the role of erm appears to be different in the embryonic versus larval Type II lineages (Curt, 2019).

In addition to the NB super-generation in B1, recent studies reveal that three different lineage topology mechanisms underlie the hyper-proliferation of the brain. First, the majority of NBs (136 out 160 NB) display a protracted phase of NB proliferation, and do not show evidence of switching from Type I to Type 0 daughter proliferation. Second, the eight MBNBs, which appear to divide in the Type I mode and never enter quiescence, also generate large lineages. Third, the 16 Type II NBs progress by budding off INP daughter cells, which divide multiple times to generate daughter cells that in turn divide once, hence resulting in lineage expansion. In contrast, in the nerve cord many NBs switch from Type I to Type 0, and all halt neurogenesis by mid-embryogenesis. The Hox anti-proliferation gradient further results in a gradient of the Type I-->0 switch and NB exit along the nerve cord. The combined effects of these alternate lineage topology behaviours translate into striking differences in the average lineage size in the brain when compared to the nerve cord. Moreover, the three different modes of more extensive NB and daughter cell proliferation combine with the super-generation of NBs in B1 to generate many more cells in the B1 brain segment, when compared to all posterior segments (Curt, 2019).

The brain TFs studied examined in this study are expressed in several or all (Tll) of the three brain NB types, and are important for both NB and daughter cell proliferation. In line with this, brain TF ectopic expression, with the late neural driver elav-Gal4, drives aberrant nerve cord proliferation and blocks both the Type I-->0 daughter cell proliferation switch and NB cell cycle exit. This results in the generation of supernumerary cells, evident both by the expansion of specific lineages and an increase in overall nerve cord cell numbers. Both the double and Tetra misexpression can trigger the ectopic generation of what appears to be a mix of Type I and Type II-like NBs. The mix of these two NB types may reflect that the misexpression scenario does not accurately and reproducibly recreate the temporal order of the brain TFs, with for example tll expressed prior to erm in the wild type (Curt, 2019).

The ectopic appearance of symmetrically dividing NBs in the brain TF co-misexpression nerve cords is more difficult to explain. However, since there normally are divisions of cells in the neuroectodermal layer prior to NB delamination, and given the early expression of the brain TFs (prior to NB delamination), it is tempting to speculate that brain TF co-misexpression to some extent can trigger an early neuroectodermal cell fate (Curt, 2019).

It was recently found that NB and daughter proliferation is also promoted by a set of early TFs expressed by most, if not all NBs. Strikingly, these TFs are expressed at higher levels in the brain, due to the lack of Hox expression therein, thereby contributing to the extended NB proliferation and more proliferative daughter cells observed in the brain. It will be interesting to address the possible regulatory interplay between these broadly expressed early NB factors and the brain TFs documented in this study (Curt, 2019).

Gene expression studies have revealed the mutually exclusive territory of brain TF and Hox gene expression in the Drosophila CNS. In line with this notion, this study found that co-misexpression of brain TFs in the nerve cord repressed expression of the posterior Hox genes of the BX-C, and conversely that BX-C co-misexpression repressed several brain TFs; Bsh, Rx, Hbn, Tll and Doc2 (Curt, 2019).

A key 'gate-keeper' of the brain versus nerve cord territories appears to be the PRC2 epigenetic complex. Removing PRC2 function results in complete loss of the H3K27me3 repressive epigenetic mark and anterior expansion of the expression of all Hox genes. This furthermore results in repression of brain TF expression, that is Tll and Doc2, as well as Rx. Surprisingly, in spite of the many roles that PRC2 may play, transgenic brain TF co-expression rescued the PRC2 mutant proliferation defects. Given the repressive action of BX-C Hox genes on brain TFs, this suggests that the principle role of PRC2 during early CNS development, at least regarding proliferation, is to ensure that Hox genes are prevented from being expressed in the brain, thus ensuring brain TF expression. Indeed, it was recently demonstrated that the reduced brain proliferation observed in esc mutants could also be fully rescued by the simultaneous removal of the posterior-most and most anti-proliferative Hox gene, Abd-B (Curt, 2019).

In mammals, the precise number of neural progenitors present at different axial levels during embryonic development has not yet been mapped. However, the wider expanse of the anterior embryonic neuroectoderm would suggest the generation of more progenitors anteriorly. There is also an extended phase of neurogenesis in the forebrain, when compared to the spinal cord. Dividing daughter cells (most often referred to as basal progenitors; bP) have been identified along the entire A-P axis of the mouse CNS. Intriguingly, the ratio of dividing bPs to apical progenitors (radial glial cells) was found to be higher in the telencephalon than in the hindbrain. Similarly, recent studies revealed a higher ratio of dividing cells in the outer layers than in the lumen, when comparing the developing telencephalon to the lumbo-sacral spinal cord. Albeit still limited in their scope, these studies suggest that a similar scenario is playing out along the A-P axis of mouse CNS as that observed in Drosophila, with an anteriorly extended phase of progenitor proliferation and a higher prevalence of proliferating daughter cells (Curt, 2019).

In addition to the similarities between Drosophila and mouse regarding progenitor generation, as well as progenitor and daughter cell proliferation, the genetic mechanisms controlling these events may also be conserved. Mouse orthologues of the Drosophila brain TFs examined in this study that is Nr2E1/Tlx (Tll); Otp, Rax and Arx (Otp); Tbx2/3/6 (Doc1/2/3); and FezF1/2 (Erm), are restricted to the brain and are known to be critical for normal mouse brain development, and in several cases for promoting proliferation. Furthermore, Hox genes are not expressed in the mouse forebrain and there is a generally conserved feature of brain TFs expressed anteriorly and Hox genes posteriorly. Mutation and misexpression has revealed that Hox genes are anti-proliferative also in the vertebrate CNS. Moreover, PRC2 (EED) mouse mutants show extensive expression of Hox genes into the forebrain and reduced gene expression of for example Nr2E1, Fezf2 and Arx. This is accompanied by reduced proliferation in the telencephalon and a microcephalic brain, while the spinal cord does not appear effected (Curt, 2019).

Gene expression and phylogenetic consideration recently led to the proposal that the CNS may have evolved by 'fusion' of two separate nervous systems, the apical and basal nervous systems, present in the common ancestor. Interestingly, in arthropods for example Drosophila, the brain and nerve cord initially form in separate regions only to merge during subsequent development. Recent studies of the role of the PRC2 complex and Hox genes in controlling A-P differences in CNS proliferation, in both Drosophila and mouse, lend support for the notion of a 'fused' CNS. This idea is further supported by recent studies of the epigenomic signature and early embryonic cell origins of the anterior versus posterior developing CNS. The findings outlined herein, showing that brain hyperproliferation is driven not only by the lack of Hox homeotic gene expression, but also by the specific expression of highly conserved brain TFs, lend further support to the notion of a separate evolutionary origin of brain and nerve cord (Curt, 2019).

It is tempting to speculate that the possibly separate evolutionary origins of the brain and nerve cord may manifest not only as distinct modes of neurogenesis, but also be reflected by separate regulatory mechanisms. These would involve brain TFs acting anteriorly, generating an abundance of progenitors, as well as driving progenitor and daughter cell proliferation. Conversely, Hox genes would act posteriorly, counteracting progenitor generation, as well as tempering progenitor and daughter cell proliferation. In this model, PRC2 would act as a 'gate keeper', ensuring that Hox genes are restricted from the brain and thereby promoting brain TF expression. This model clearly represents an over-simplification, but may serve as a useful launching point for future comparative studies in many model systems (Curt, 2019).

REGULATION

Promoter

HLH54F, the Drosophila ortholog of the vertebrate basic helix-loop-helix domain-encoding genes capsulin and musculin, is expressed in the founder cells and developing muscle fibers of the longitudinal midgut muscles. These cells descend from the posterior-most portion of the mesoderm, termed the caudal visceral mesoderm (CVM), and migrate onto the trunk visceral mesoderm prior to undergoing myoblast fusion and muscle fiber formation. HLH54F expression in the CVM is regulated by a combination of terminal patterning genes and snail. HLH54F mutations were generated and this gene was shown to be crucial for the specification, migration and survival of the CVM cells and the longitudinal midgut muscle founders. HLH54F mutant embryos, larvae, and adults lack all longitudinal midgut muscles, which causes defects in gut morphology and integrity. The function of HLH54F as a direct activator of gene expression is exemplified by analysis of a CVM-specific enhancer from the Dorsocross locus, which requires combined inputs from HLH54F and Biniou in a feed-forward fashion. It is concluded that HLH54F is the earliest specific regulator of CVM development and that it plays a pivotal role in all major aspects of development and differentiation of this largely twist-independent population of mesodermal cells (Ismat, 2010).

The Drosophila mesoderm forms from the ventral-most cells of the early embryo that invaginate during gastrulation. The expression and function of two transcription factors, the basic helix-loop-helix (bHLH) protein Twist (Twi) and the zinc-finger protein Snail (Sna), in ventral cells located between ~15% and 85% egg length are essential for their invagination and for subsequent mesodermal tissue development. Although the absence of either twi or sna activity results in similar phenotypes, the molecular roles of the two genes in this pathway differ. twi functions in activating a variety of mesoderm-specific target genes, including several that are known to regulate the invagination, patterning and differentiation of the mesoderm. By contrast, sna is thought to act largely, if not exclusively, as a repressor of a number of neuroectodermal targets, permitting mesoderm formation by restricting the expression of these genes to areas outside the presumptive mesoderm (Ismat, 2010).

Notably, however, there is at least one group of mesodermal cells that requires sna, but not twi, for its initial phase of development. This cell group is located ventrally within the domain of early twi and sna expression, but is restricted to the posterior tip of the mesoderm between ~7.5% and 15% egg length. Because it is fated to develop into the longitudinal muscles of the midgut, it has been termed the caudal visceral mesoderm (CVM) primordium. In light of the apparent lack of a requirement for twi for the initial development of the CVM, it is interesting that the cells of the CVM primordium are marked by the expression of another bHLH-encoding gene, HLH54F. This situation raises the possibility that, in the caudal-most portion of the mesoderm, HLH54F instead of twi cooperates with sna to control early CVM development. However, until now, specific mutants for HLH54F have not been available to test this possibility (Ismat, 2010).

The Drosophila midgut musculature consists of syncytial fibers that arise through myoblast fusion between gut muscle founder cells and fusion-competent myoblasts and forms a meshwork of circular and longitudinal muscles around the endodermal layer. The CVM appears to be the sole source of founder cells of the longitudinal midgut muscles. After their ingression, these cells migrate anteriorly and spread over the future midgut, where they fuse with resident fusion-competent cells to form the multinucleated longitudinal muscle fibers of the midgut. The fusion-competent cells for this event come from a different source, the so-called trunk visceral mesoderm (TVM), which provides a second (and major) contribution of precursors to the musculature of the midgut. The primordia of the TVM are arranged bilaterally as 11 metameric cell clusters within the dorsal mesoderm, which subsequently merge with each other into the contiguous band of the TVM. The TVM primordia are marked by the expression of the NK homeodomain gene bagpipe (bap) and the FoxF gene biniou (bin), both of which are essential for TVM formation. Within the TVM, the founder cells of the circular midgut muscles are induced by Jelly belly (Jeb) signals acting through the receptor tyrosine kinase Alk. The founder myoblasts from the TVM fuse one-to-one with adjacent fusion-competent myoblasts into binucleated syncytia that form the circular midgut muscles. Subsequently, after the migrating CVM-derived founder cells have arrived at their destinations, each fuses with multiple fusion-competent cells from the TVM left over from the TVM founder cell fusions. It is these multinucleated syncytia that will then differentiate into the longitudinal visceral muscles, which run perpendicularly to the circular muscles along the entire length of the midgut. The longitudinal gut muscle fibers from the outer layer of the developing visceral musculature are tightly interwoven with the circular gut muscle fibers from the inner layer (Ismat, 2010).

HLH54F is the earliest known marker of the CVM primordia and its expression is maintained throughout the development and differentiation of the longitudinal midgut muscles. To test whether HLH54F plays an important role in the development of the CVM and longitudinal midgut musculature, loss-of-function mutations for this gene were generated by imprecise P-excision and EMS mutagenesis screens. This study demonstrates that in the absence of HLH54F activity, no longitudinal gut muscle founder cells are formed. The absence of all tested CVM markers and the observed apoptotic death of the cells that would normally be destined to form CVM in HLH54F mutants, show that HLH54F has an essential role in determining the CVM and in specifying the founder cells of the longitudinal gut musculature. This function includes feed-forward regulation and direct binding to target enhancers (e.g., from the Dorsocross genes). It was also shown that ectopic expression of HLH54F can interfere with normal somatic muscle, cardiac and TVM development. Further, the known pathway of CVM development has been extended by showing that the initiation of HLH54F expression is largely independent of twi, but depends critically on the combined activities of sna and terminal patterning genes, particularly the synergistic activities of fork head (fkh) and brachyenteron (byn). Hence, the CVM primordia are determined at the intersection of the domains of these mesodermal and terminal regulators (Ismat, 2010).

This study has shown that HLH54F is a key regulator in the CVM, a population of cells in which the bHLH gene twi appears to have only minor functions. Although twi is initially co-expressed with HLH54F in these cells, it makes only a small contribution to activating HLH54F expression, and the expression of both bHLH genes rapidly becomes mutually exclusive. Instead of twi, the activation of HLH54F in the CVM primarily involves the combined activities of mesodermal sna and the terminal genes fkh and byn. As sna is generally thought to act as a ventral repressor of non-mesodermal genes in early mesoderm development, it will be interesting to determine whether the positive requirement for sna in the activation of HLH54F expression is direct, which would be unique to date. Alternatively, HLH54F might be activated by high levels of nuclear Dorsal and repressed by lateral genes that are repressed by sna ventrally. Along the anteroposterior axis, the posterior border of HLH54F expression is apparently defined by the posterior expression border of sna, which is delineated by the repressive action from hkb. It is proposed that the anterior border of HLH54F is determined by near-maximal threshold levels of tll, the expression of which declines steeply in the area anterior to the HLH54F domain. However, tll acts largely indirectly, through the combined activities of its downstream genes byn and fkh, in activating HLH54F. The low residual levels of HLH54F mRNA in fkh byn double mutants suggest the involvement of direct inputs from additional posterior activities, possibly tll or maternal torso. It appears that high-level expression of zfh1 in the CVM largely depends on tll and sna, whereas HLH54F and zfh1 do not depend on one another (Ismat, 2010).

Notably, neither twi nor HLH54F is required individually for the internalization of the CVM cells during gastrulation, although a redundant function cannot be excluded. The posterior portion of the mesoderm, which includes the CVM and portions of the presumptive HVM, bends around during gastrulation to form a second, internal mesodermal layer. It is conceivable that this movement is a passive process brought about by the invagination of the PMG rudiment. However, for subsequent migrations of CVM cells from these positions, the activity of HLH54F, but not twi, is crucial. In addition, byn, zfh1 and fkh are required for normal migration after stage 10. Whereas their respective functions are likely to be cell-autonomous, the observed requirement of twi for normal pathfinding of CVM cells is likely to be due to the absence of the migration substrate normally formed from the trunk mesoderm (Ismat, 2010).

The genetic data show that, after the caudal mesodermal cells have ingressed in this manner, they do not develop any further in the absence of HLH54F activity and undergo apoptosis. In the normal situation, HLH54F is needed for the activation of several transcription factor-encoding genes at this stage, including bin, croc and the Doc genes. Although the functions of these genes in CVM development have not been defined, it is likely that they regulate specific aspects of CVM development downstream of, and perhaps in combination with, HLH54F. The data from loss-of-function and ectopic expression analyses of HLH54F show that this gene is essential, but not sufficient, for specification of longitudinal gut muscle founders. Parallel inputs, albeit less pervasive, appear to come from high-level zfh1, which like HLH54F is required for croc/croc-lacZ expression. Altogether, it is proposed that HLH54F is necessary for activating the vast majority of early CVM-specific genes, with one known exception being high-level zfh1, and that zfh1, byn and fkh in various combinations act together with HLH54F to activate certain targets during the specification and early migration of CVM cells (Ismat, 2010).

The continuous expression of HLH54F in the CVM and longitudinal gut muscles suggests that this gene is not only required for specification, but is also directly involved in many other developmental processes, including the continued migration, myoblast fusion and differentiation of the CVM cells. Possible downstream targets of HLH54F in the promotion of proper cell migration include beat-IIa, which encodes an as yet uncharacterized membrane-anchored Ig domain protein, and the FGF receptor-encoding gene heartless (htl), which is known to be required for normal migration. In this context, it is interesting that the vertebrate orthologs of HLH54F are expressed prominently in specific migrating populations of mesodermal cells as well. For example, musculin is expressed in myoblasts at the myotomal lips that migrate into the developing limbs, and capsulin is expressed in the migrating pro-epicardial cells . Therefore, it is possible that parts of the regulatory circuit in the control of cell migration have been conserved, even though they occur in different mesodermal cell types. capsulin is also expressed prominently in the splanchnopleura and tissues derived from it, including the developing smooth muscles of the stomach and gut. Therefore, HLH54F and capsulin might share some functions in the terminal differentiation of the respective gut musculatures in the different systems. Both Capsulin and Musculin have been characterized largely as repressors. However, their activity (and likewise that of HLH54F) as repressors versus activators might well be context specific with respect to the particular enhancer, tissue or developmental stage in question and might depend on the relative concentrations of particular heterodimerization partners (Ismat, 2010).

Based on the phenotype of byn mutants, a role of the CVM in promoting midgut constrictions has also been proposed. The phenotype of HLH54F mutants confirms this effect, although it was found that partial constrictions can frequently occur and that the effect is variable. It is inferred that the physical interactions between developing longitudinal and circular muscle fibers are necessary to provide the full force required for the efficient constriction of the midgut endoderm at the well-defined signaling centers. In the fully developed midgut, scanning electron microscopy images have revealed that the longitudinal fibers are tightly interwoven with the web-shaped circular fibers, which may explain the mechanical strength of this meshwork. Indeed, it was found that the mechanical stability and integrity of the midgut, particularly in HLH54F mutant adults, are severely compromised (Ismat, 2010).

In summary, HLH54F appears to sit at the top of the regulatory hierarchy of CVM development and is likely to fulfill additional key roles during the course of development of the CVM and the longitudinal gut muscles. Future efforts need to be directed towards dissecting additional downstream events that regulate the different steps of cell migration, myoblast fusion, morphogenesis and terminal differentiation of the longitudinal midgut musculature (Ismat, 2010).

Transcriptional Regulation

Since peak levels of Dpp activity are known to be required for cell fate determination at the dorsal midline, the correlation between Dpp activity and dorsal longitudinal Doc expression during blastoderm stages was examined. Double-staining for Doc mRNA and phosphorylated Mad (PMad) indicates a close correlation between cells containing high levels of PMad and Doc products within the dorsal-longitudinal stripe. In addition, faint Doc signals that are modulated in a pair-rule pattern extend into areas that receive lower Dpp inputs and lack detectable PMad. Both PMad and Doc expression in the dorsal-longitudinal stripe, but not the dorsal-transverse head stripe of Doc expression, are absent in dpp-null mutant embryos. Conversely, in blastoderm embryos with ubiquitous Dpp expression (UAS-dpp activated by maternally provided nanos-GAL4), a significant widening is observed of the dorsal-longitudinal stripes of PMad and Doc expression, during which the correlation between high PMad and Doc mRNA levels is still maintained (Reim, 2003).

The expansion of PMad upon uniform ectopic expression of dpp includes the prospective mesoderm, although not ventrolateral areas of the blastoderm embryo. However, high PMad in the prospective mesoderm does not trigger ectopic Doc expression, suggesting either the presence of a ventral repressor or the requirement for a co-activator in dorsal areas. A candidate for a co-activator is the homeobox gene zerknüllt (zen). Double in situ hybridization shows that the appearance of dorsal Doc mRNAs coincides with the time when zen mRNA levels increase in the areas of the presumptive amnioserosa as a result of high Dpp inputs. When the refinement of zen expression is completed, there is an exact correspondence in the widths of the Doc and zen expression domains -- although Doc expression extends more posteriorly, the activity of zen is necessary for normal levels of Doc expression in the dorsal-longitudinal stripe, because in zen mutant embryos there are only low residual levels of Doc products present in this domain. These observations suggest that Doc expression along the dorsal midline of blastoderm embryos requires the combined activities of dpp and zen (Reim, 2003).

The known distribution of dpp mRNA during its second phase of expression in the dorsolateral ectoderm of stage 9-11 embryos suggests that Doc expression in the dorsolateral ectoderm and mesoderm during these stages is also dependent on Dpp activity. As expected from the known fate map shifts in dpp mutants, these domains of Doc expression are missing in dpp-null mutant embryos. Notably, the exact coincidence between the ventral borders of the domains of dorsolateral Doc expression and high nuclear PMad suggests that Doc expression is directly controlled by Dpp-activated Smad proteins in the ectoderm and mesoderm during this stage. Additional evidence for this hypothesis comes from experiments with ectopic expression of dpp in the ventral ectoderm of the Krüppel domain (by virtue of a modified Kr-GAL4 driver), that results in the concomitant expansion of PMad and the Doc expression stripes towards the ventral midline (Reim, 2003).

In addition to the inputs from dpp, metameric Doc expression in dorsolateral areas of the germ band must depend on the activity of segmental regulators. A direct comparison with the expression of engrailed (en) shows that the clusters of Doc expression straddle the compartmental borders. Although Doc expression overlaps with en in the P compartments, about two-thirds of the Doc expressing cells of each cluster are located in posterior areas of the A compartments. In agreement with this allocation, it has been found that the metameric Doc domains are exactly centered on the stripes of Wingless (Wg) expression. The observed correlation of the segmental registers of Wg and Doc makes wg a good candidate for an upstream regulator of Doc. Dorsolateral Doc expression in the ectoderm and mesoderm is shown to be completely absent if wg is inactive. By contrast, deletion of sloppy paired (slp), a known target of wg in the mesoderm and a wg feedback regulator in the ectoderm, results in a reduction, but not a complete loss of metameric DOC expression. Hence, slp probably affects Doc indirectly through its effect on ectodermal wg expression. Altogether, the data suggest that metameric Doc expression in the ectoderm and mesoderm is triggered by the intersecting activities of Wg and Dpp (Reim, 2003).

Targets of Activity

The segmental stripes of wg expression in the embryonic trunk segments initially span the entire dorsoventral extent of the ectoderm, but at stage 11 they become interrupted in dorsolateral areas. A comparison of Wg and Doc expression at this stage shows that the positions of the metameric ectodermal domains of Doc expression correspond to the areas in which the Wg stripes become interrupted. Temporally, there is a brief overlap of ectodermal Wg and Doc expression during stage 10 until Wg expression is downregulated within the Doc domains. In contrast to the wild-type situation, the Wg stripes remain continuous in DocA mutant embryos. Similar observations were made with the homeobox gene product Ladybird (Lb=Lbe + Lbl) as a marker. In wild-type embryos after stage 11, Lb is also expressed in striped domains that are interrupted at the positions of the ectodermal Doc domains, whereas in DocA mutant embryos there is ectopic expression in a pattern of continuous stripes. These data show that Doc activity is required for patterning events in the dorsolateral ectoderm, which include the repression of wg and lb expression in these areas (Reim, 2003).

Ectopic expression experiments with Doc genes provide additional evidence for a repressive activity of Doc on wg expression. Upon ectopic expression of Doc2 in all cells of the ectoderm of wild type embryos, the ventral portions of the Wg stripes are lost. However, the dorsal regions of the Wg stripes appear to be under different regulation, because ectopic Doc results in a uniform domain of dorsal Wg along the anteroposterior axis, albeit at lower levels than in wild-type embryos (Reim, 2003).

Ectopic expression experiments with Doc genes in imaginal discs further confirm their ability to repress wg. In third instar larval wing discs, Doc genes are expressed in four distinct areas that do not overlap with the wg expression domains. Specifically, two large Doc expression domains are located in the centers of the dorsal and ventral regions of the prospective wing blades and two smaller domains in prospective dorsal hinge and posterior notal regions, respectively. In leg discs, low levels of Doc expression can be detected in regions of the prospective body wall and proximal leg segments, which also do not express wg. Importantly, ectopic expression of Doc2 within the Dpp domains of imaginal discs causes wg expression to disappear in the corresponding areas. In agreement with the known role of wg in limb development, its repression by ectopic Doc results in the loss of distal structures of wings, legs and antenna of adult animals. Analogous ectopic expression experiments with Doc1 and Doc3 in embryos and discs produce qualitatively similar (although weaker) effects to those of Doc2 (Reim, 2003).

Expression, regulation, and requirement of the Toll transmembrane protein during dorsal vessel formation; The Toll transcriptional enhancer is regulated by both Doc and Tin

Early heart development in Drosophila and vertebrates involves the specification of cardiac precursor cells within paired progenitor fields, followed by their movement into a linear heart tube structure. The latter process requires coordinated cell interactions, migration, and differentiation as the primitive heart develops toward status as a functional organ. In the Drosophila embryo, cardioblasts emerge from bilateral dorsal mesoderm primordia, followed by alignment as rows of cells that meet at the midline and morph into a dorsal vessel. Genes that function in coordinating cardioblast organization, migration, and assembly are integral to heart development, and their encoded proteins need to be understood as to their roles in this vital morphogenetic process. The Toll transmembrane protein is expressed in a secondary phase of heart formation, at lateral cardioblast surfaces as they align, migrate to the midline, and form the linear tube. The Toll dorsal vessel enhancer has been characterized, with its activity controlled by Dorsocross and Tinman transcription factors. Consistent with the observed protein expression pattern, phenotype analyses demonstrate Toll function is essential for normal dorsal vessel formation. Such findings implicate Toll as a critical cell adhesion molecule in the alignment and migration of cardioblasts during dorsal vessel morphogenesis (Wang, 2005).

At the time dorsal-ventral polarity is established during early Drosophila development, Toll is associated with the plasma membrane around the entire syncytial blastoderm embryo. Thereafter, Toll exhibits zygotic expression on several cell surfaces, including a specific dorsal cell type in late-stage embryos. These were identified at first as leading-edge cells of the two-epidermal sheets moving toward the dorsal midline. Toll expression in dorsal aspects of the embryo has been reevaluated and, to the contrary, it has now been concluded the gene is expressed in cardioblasts of the developing and formed dorsal vessel (Wang, 2005).

Initially, Toll mRNA accumulation was analyzed by in situ hybridization, with gene transcripts first detected in dorsal cell populations in stage 12 embryos and later in two converging rows of cells during the process of dorsal closure. The likelihood of the Toll-positive cells being cardioblasts was strongly implied by the pattern of mRNA accumulation in stage 16 embryos. Toll expression was detected in roughly 50 cell pairs, and the organization of said cells was reminiscent of cardioblasts within structurally identifiable aorta and heart regions of the assembled dorsal vessel. The pattern of Toll protein expression was also investigated, with results comparable to those obtained in the RNA analysis. The transmembrane protein was detected in dorsal cells in late stage 12/early stage 13 embryos. Thereafter, it showed a clear presence on lateral surfaces of all cells aligned within two contiguous rows as they migrate toward the dorsal midline. By stage 16, the Toll-positive cells populate the core of the dorsal vessel, again within defined aorta and heart subregions. Toll was found exclusively on cardioblast surfaces, while organ-associated pericardial, lymph gland, and ring gland cells failed to express the protein. High-resolution analysis by confocal microscopy demonstrated Toll presence at lateral points of contact between all cardioblasts of the mature dorsal vessel (Wang, 2005).

Toll zygotic transcription is complex based on the numerous cell and tissue types that express the gene. Through efforts to identify a regulatory sequence controlling Toll expression in central nervous system (CNS) midline glial cells, Wharton (1993) located three regions upstream of the gene that possessed transcriptional enhancer activity. Relevant to the demonstration of Toll expression in the dorsal vessel, a 6.5-kb DNA was fortuitously found to direct lacZ reporter expression in all cardioblasts, and in pharyngeal and body wall muscles as well. Due to the interest in understanding how this expression might be regulated, the Toll cardioblast enhancer was delimited within the defined upstream region. At first, the analysis involved testing Toll 5'-flanking DNAs for the ability to drive lacZ expression in embryos of transgenic strains. A 7.1-kb region located between ~9.3 and ~2.2 upstream of the gene showed strong enhancer function in all cardioblasts of the dorsal vessel. The DNA was subdivided into five overlapping segments, and only the most distal 1.7-kb DNA maintained cardioblast activity. Subsequently, five fragments spanning this 1.7-kb interval were tested for enhancer function, and dorsal vessel activity was mapped to a 305-bp sequence located between ~8.3 and ~8.0 relative to the Toll transcription start site. Consistent with the timing of Toll mRNA and protein accumulation in cardioblasts, the 305-bp enhancer becomes active during stage 12 and maintains its activity through all subsequent events of dorsal vessel morphogenesis. It is noteworthy that this small DNA also functions in amnioserosa cells from stage 11 through stage 15 (Wang, 2005).

Since Toll encodes a transmembrane protein with leucine-rich repeats in its extracellular domain, a prediction was made that Toll could function as a homophilic cell adhesion molecule, in addition to its well-characterized role as a signal-transducing receptor. In support of this hypothesis, induced expression of the protein in the nonadhesive Schneider 2 cell line causes cellular aggregation, with Toll accumulating at sites of cell-cell interaction. Such a localization property is characteristic of cellular adhesion molecules. Given the highly specialized localization, and structural and functional features of the protein, it is likely that Toll contributes prominently to the molecular environment that aligns and stabilizes cardioblasts on their path toward assembly within the dorsal vessel (Wang, 2005).

The observation of structurally defective dorsal vessels within Toll mutant embryos is consistent with the pattern of Toll expression in cardioblasts. D-MEF2 serves as a marker for all cardioblasts, from their early appearance through their organization within the mature organ. Based on D-MEF2 staining, it appears appropriate numbers of cardioblasts are specified in mutant embryos, but deviations are observed from the normal process of cardioblast alignment and synchronous migration as two contiguous rows of 52 cells. Several other markers for the formed dorsal vessel identified random gaps in the linear organ due to missing and/or abnormally located cardioblasts. Such cardiac phenotypes are reminiscent of those presented by faint sausage (fas) mutant embryos; mutations of the immunoglobulin-like cell adhesion molecule also led to cardioblast alignment problems. Whether Toll and Fas work in combination for the proper alignment and migration of these cells remains to be investigated. Additionally, while structural and phenotypic properties are consistent with its role as a cardioblast adhesion molecule, a function for Toll in mediating signaling events between neighboring cardiac cells cannot be ruled out. So far, no indicators exist for the latter possibility; it was not possible to demonstrate expression of potential Toll transcriptional effectors (Dorsal and Dif) in cells of the dorsal vessel. Either way, these molecular and genetic findings identify Toll as a vital player in dorsal vessel formation (Wang, 2005).

The regulation of Toll expression in cardioblasts was pursued due to an interest in further defining the transcriptional network controlling heart development in Drosophila. The studies demonstrated Toll heart expression is controlled by a 305-bp DNA located 8.0 kb upstream of the transcription start site. This regulatory module contains multiple binding sites for Doc T-box proteins and a single recognition site for the Tin homeodomain protein. The Toll dorsal vessel enhancer contains a single TCAAGTG sequence at nucleotides 163 to 169. The evidence is strong for the transcriptional enhancer being regulated by both of these cardiogenic factors. Doc and Tin are expressed in adjacent but nonoverlapping sets of cardioblasts within segments of the dorsal vessel; together, they make up the complete population of inner cardiac cells. A deletion of the distal part of the Toll 305-bp enhancer that removes the strong Doc-A footprint sequence, which likely binds multiple Doc molecules through T-box domain recognition of GTG motifs, eliminates enhancer function in Svp/Doc cells while maintaining activity in Tin cells. Systematically adding back T-box core binding elements to partially, then fully, reestablish the Doc-A binding site restores enhancer function in the Svp/Doc population (Wang, 2005).

As for Tin, mutation of its recognition element in the Toll 305 DNA leads to decreased and variable enhancer activity in both Tin and Svp/Doc cardioblasts. This result suggests that Tin is required not only for the activation of Toll expression in the four cardioblasts per hemisegment that are Tin positive after stage 12 but also for its initiation in all six cardioblasts in each hemisegment during early stage 12. The residual activity of the mutated Toll 305 DNA may reflect some degree of Tin regulation through cryptic, low-affinity binding sites present in the enhancer. Indeed, perusal of the Toll sequence identifies three candidate Tin elements that match the binding consensus at six of seven nucleotide pairs, and other Tin-regulated enhancers of genes such as D-mef2, ß3-tubulin, and pnr also employ more than one Tin binding site (Wang, 2005).

In contrast to the Toll 305 enhancer element, mutation of the exact Tin site in the Toll 258 DNA completely silenced the enhancer in the normally Tin-active cells. This result strongly implies that Tin, and at least one other factor working through the distal 47 bp of DNA, are required for activating the Toll gene. Candidates for such factors are the Doc T-box proteins, which are initially expressed in all cardioblast progenitors during mid stage 12, as well as products of the T-box genes H15 and Midline (Mid), which are expressed in all cardioblasts from mid stage 12 onward. Mid can bind to the same regions of Toll DNA as Doc, although the relevance of such interactions remains to be investigated. A combinatorial requirement for T-box proteins and Tin during the initiation and/or maintenance of Toll expression is further supported by the observation that derivatives of the enhancer containing only the Doc-A sequences fail to show activity in Svp/Doc cells. Together, these molecular data point to a mechanism wherein T-box proteins, in combination with Tin, initially activate the Toll gene in all cardioblast progenitors. After stage 12, Doc and Tin (perhaps in cooperation with H15 and/or Mid) activate Toll in two complementary subsets of cardioblasts of the dorsal vessel (Wang, 2005).

Unfortunately, a genetic requirement for these two factors in the regulation of the Toll enhancer cannot be proven at this time since Doc and tin mutant embryos fail to produce cardioblasts. Such an analysis could be attempted with the generation of specialized Doc or tin genetic backgrounds that allow for cardioblast specification early on, while lacking protein functions in later stages of dorsal vessel formation. However, forced-expression studies have demonstrated that individual expression of Tin or Doc2 leads to expanded enhancer activity, while simultaneous expression of the cardiac factors results in a robust activation of Toll transcription. These findings convincingly support the model of Doc and Tin being positive transcriptional regulators of the Toll dorsal vessel enhancer (Wang, 2005).

In addition to the demonstration of Doc and Tin as activators of Toll expression in the dorsal vessel, the regulatory analysis has generated important reagents that should facilitate the discovery of novel cardiac-functioning genes of Drosophila. That is, the Toll-cGFP and Toll-nGFP transgenes serve as sensitive markers for assessing distinct aspects of dorsal vessel morphogenesis in living animals. In stage 16 to 17 embryos and thereafter, Toll-cGFP expression can be used to monitor the formation and function of the three pairs of valvelike ostia within the heart region of the dorsal vessel. Likewise, Toll-nGFP can be used to determine the exact number and diversification status of cardioblasts, as larger nuclei are present within Tin-determined cells while smaller nuclei are found in Svp/Doc-determined cells. Such sensitive and easy-to-use reagents will be valuable in genomewide screens to discover new genes involved in Drosophila heart development (Wang, 2005).

A transcription factor collective defines cardiac cell fate and reflects lineage history

Cell fate decisions are driven through the integration of inductive signals and tissue-specific transcription factors (TFs), although the details on how this information converges in cis remain unclear. This study demonstrates that the five genetic components essential for cardiac specification in Drosophila, including the effectors of Wg and Dpp signaling, act as a collective unit to cooperatively regulate heart enhancer activity, both in vivo and in vitro. Their combinatorial binding does not require any specific motif orientation or spacing, suggesting an alternative mode of enhancer function whereby cooperative activity occurs with extensive motif flexibility. A fraction of enhancers co-occupied by cardiogenic TFs had unexpected activity in the neighboring visceral mesoderm but could be rendered active in heart through single-site mutations. Given that cardiac and visceral cells are both derived from the dorsal mesoderm, this 'dormant' TF binding signature may represent a molecular footprint of these cells' developmental lineage (Junion, 2012).

Dissecting transcriptional networks in the context of embryonic development is inherently difficult due to the multicellularity of the system and the fact that most essential developmental regulators have pleiotropic effects, acting in separate and sometimes interconnected networks. This study presents a comprehensive systematic dissection of the cis-regulatory properties leading to cardiac specification within the context of a developing embryo. The resulting compendium of TF binding signatures, in addition to extensive in vivo and in vitro analysis of enhancer activity, revealed a number of insights into the regulatory complexity of developmental programs (Junion, 2012).

Nkx (Tinman in Drosophila), GATA (Pannier in Drosophila), and T box factors (Doc in Drosophila) regulate each other’s expression in both flies and mice, where they form a recursively wired transcriptional circuit that acts cooperatively at a genetic level to regulate heart development across a broad range of organisms. The data demonstrate that this cooperative regulation extends beyond the ability of these TFs to regulate each other’s expression. All five cardiogenic TFs (including dTCF and pMad) converge as a collective unit on a very extensive set of mesodermal enhancer elements in vivo (Tin-bound regions) and also in vitro (in DmD8 cells). Importantly, this TF co-occupancy occurs in cis, rather than being mediated via crosslinking of DNA-looping interactions bringing together distant sites. Examining enhancer activity out of context, for example, in transgenic experiments and luciferase assays, revealed that the TF collective activity is preserved in situations in which these regions are removed from their native genomic 'looping' context (Junion, 2012).

In keeping with the conserved essential role of these factors for heart development, the integration of their activity at shared enhancer elements may also be conserved. Recent analyses of the mouse homologs of these TFs (with the exception of the inductive signals from Wg and Dpp signaling) in a cardiomyocyte cell line support this, revealing a signifcant overlap in their binding signatures (He, 2011; Schlesinger, 2011), although interestingly not in the collective 'all-or-none' fashion observed in Drosophila embryos. This difference may result from the partial overlap of the TFs examined, interspecies differences, or the inherent differences between the in vivo versus in vitro models. Examining enhancer output for a large number of regions indicates that this collective TF occupancy signature is generally predictive of enhancer activity in cardiac mesoderm or its neighboring cell population, the visceral mesoderm—expression patterns that cannot be obtained from any one of these TFs alone (Junion, 2012).

There are currently two prevailing models of how enhancers function. The enhanceosome model suggests that TFs bind to enhancers in a cooperative manner directed by a specific arrangement of motifs, often having a very rigid motif grammar. An alternative, the billboard model, suggests that each TF (or submodule) is recruited independently via its own sequence motif, and therefore the motif spacing and relative orientation have little importance. The results of this study indicate that cardiogenic TFs are corecruited and activate enhancers in a cooperative manner, but this cooperativity occurs with little or no apparent motif grammar to such an extent that the motifs for some factors do not always need to be present. This is at odds with either the enhanceosome (cooperative binding; rigid grammar) or billboard (independent binding; little grammar) models and represents an alternative mode of enhancer activity, which was termed a 'TF collective' (cooperative binding; no grammar), and likely constitutes a common principle in other systems (Junion, 2012).

The data suggest that the TF collective operates via the cooperative recruitment of a large number of TFs (in this case, at least five), which is mediated by the presence of high-affinity TF motifs for a subset of factors initiating the recruitment of all TFs. The occupancy of any remaining factor(s) is most likely facilitated via protein-protein interactions or cooperativity at a higher level such as, for example, via the chromatin activators CBP/ p300, which interact with mammalian GATA and Mad homologs. This model allows for extensive motif turnover without any obvious effect on enhancer activity, consistent with what has been observed in vivo for the Drosophila spa enhancer and mouse heart enhancers (Junion, 2012).

Integrating the TF occupancy data for all seven major TFs involved in dorsal mesoderm specification (the five cardiogenic factors together with Biniou and Slp) revealed a very striking observation: the developmental history of cardiac cells is reflected in their TF occupancy patterns. Visceral mesoderm (VM) and cardiac mesoderm (CM) are both derived from precursor cells within the dorsal mesoderm. Once specified, these cell types express divergent sets of TFs: Slp, activated dTCF, Doc, and Pnr function in cardiac cells, whereas Biniou and Bagpipe are active in the VM. Despite these mutually exclusive expression patterns, the cardiogenic TFs are recruited to the same enhancers as VM TFs in the juxtaposed cardiac mesoderm. Moreover, dependent on the removal of a transcriptional repressor, these combined binding signatures have the capacity to drive expression in either cell type. This finding provides the exciting possibility that dormant TF occupancy could be used to trace the developmental origins of a cell lineage. It also explains why active repression in cis is required for correct lineage specification, which is a frequent observation from genetic studies. At the molecular level, it remains an open question why the VM-specific enhancers are occupied by the cardiac TF collective. It is hypothesized that this may occur through chromatin remodeling in the precursor cell population. An 'open' (accessible) chromatin state at these loci in dorsal mesoderm cells, which is most likely mediated or maintained by Tin binding prior to specification, could facilitate the occupancy of cell type-specific TFs in both CM and VM cells. Such early 'chromatin priming' of regulatory regions active at later stages has been observed during ES cell differentiation. The current data provide evidence that this also holds true for TF occupancy and not just chromatin marks. On a more speculative level, this developmental footprint of TF occupancy may reflect the evolutionary ancestry of these two organs. Visceral and cardiogenic tissues are derived from the splanchnic mesoderm in both flies and vertebrates. These complex VM-heart enhancers may represent evolutionary relics containing functional binding sites that reflect enhancer activity in an ancestral cell type (Junion, 2012).

Taken together, the collective TF occupancy on enhancers during dorsal mesoderm specification illustrates how the regulatory input of cooperative TFs is integrated in cis, in the absence of any strict motif grammar. This more flexible mode of cooperative cis regulation is expected to be present in many other complex developmental systems (Junion, 2012).

Dorsoventral patterning of the Drosophila hindgut is determined by interaction of genes under the control of two independent gene regulatory systems, the dorsal and terminal systems

Dorsoventral (DV) patterning in the trunk region of Drosophila embryo is established through intricate molecular interactions that regulate Dpp/Scw signaling during the early blastoderm stages. The hindgut of Drosophila, which derives from posterior region of the cellular blastoderm, also shows dorsoventral patterning, being subdivided into distinct dorsal and ventral domains. engrailed (en) is expressed in the dorsal domain, which determines dorsal fate of the hindgut. This study shows that a repressor Brk restricts en expression to the dorsal domain of the hindgut. Expression domain of brk during early blastodermal stages is defined through antagonistic interaction with dpp, and expression domains of dpp and brk in the early blastoderm include prospective hindgut domain. After stage 9, dpp expression in the dorsal domain of the hindgut primordium disappears, but, the brk expression in the ventral domain continues. It was found that Dorsocross (Doc), which is a target gene of Dpp, is responsible for restricting brk expression to the ventral domain of the hindgut. On the other hand, activation of en is under the control of brachyenteron (byn) that is regulated independently of dpp, brk, and Doc. The cooperative interaction of common DV positional cues with byn during hindgut development represents another aspect of mechanisms of DV patterning in the Drosophila embryo (Hamaguchi, 2012).

A model is presented of the genetic pathway leading to the DV subdivision of the Drosophila hindgut. The dorsal fate of the hindgut is finally determined by a selector gene en that is expressed in the dorsal domain of the hindgut. The present study revealed that DV patterning of the hindgut is based on antagonistic interaction of Dpp and Brk in the early cellular blastoderm. Dpp expression disappears in the hindgut primordium after stage 9, but, Dpp target gene Doc takes over the repressive effect on Brk, and restricts Brk expression to the ventral domain. In other words, primary role of Dpp in the hindgut development is to activate Doc genes in the dorsal domain for repression of Brk. Eventually, Brk represses the selector gene en in the ventral domain, restricting it to the dorsal domain. This is the outline of gene regulatory pathway of DV patterning of the hindgut. It should be noted that Doc represses brk in the dorsal domain, while Brk does not regulate Doc expression in the hindgut in normal development, since brk mutation does not affect Doc expression in the hindgut. Dpp and Doc do not determine the dorsal fate directly, but, act indirectly by repressing brk in the dorsal domain. In fact, brk; DocA (null) double-mutant embryos, as well as brk; dpp double mutant embryos, expressed en in both dorsal and ventral domains of the hindgut. This regulatory interrelation between brk and Dpp/Doc is partially reminiscent of that in the wing discs, in which primary role of Dpp is repression of brk, and the latter is responsible for defining antero-posterior pattern of gene expression in the wing discs. Repression of brk by Dpp signals has been reported to depend on the zinc finger protein Schnurri (Shn) in some tissues. However, brk expression in the hindgut did not expand dorsally in the shn mutant embryo, and also, en expression was observed in the hindgut in shn mutant embryo. Thus, shn may not be essential for regulation of brk in the hindgut (Hamaguchi, 2012).

However, activation of en in the hindgut, is under the control of byn, the process of which is independent of DV patterning. The expression domain of byn is included in the region where intricate interaction of dpp, brk, and Doc proceeds. The dpp, brk, and Doc genes are all under the control of the dorsal system, while byn is activated under the control of the terminal system that provides AP positional cues in terminal regions of the early blastoderm. In other words, AP positional cues activate en, while DV positional cues repress en. Thus, cooperative interaction of the two independent gene regulatory systems establishes the expression pattern of en in the hindgut (Hamaguchi, 2012).

Protein Interactions

Drosophila Groucho, like its vertebrate Transducin-like Enhancer-of-split homologues, is a corepressor that silences gene expression in numerous developmental settings. Groucho itself does not bind DNA but is recruited to target promoters by associating with a large number of DNA-binding negative transcriptional regulators. These repressors tether Groucho via short conserved polypeptide sequences, of which two have been defined: (1) WRPW and related tetrapeptide motifs have been well characterized in several repressors; (2) a motif termed Engrailed homology 1 (eh1) has been found predominantly in homeodomain-containing transcription factors. A yeast two-hybrid screen is described that uncovered physical interactions between Groucho and transcription factors, containing eh1 motifs, with different types of DNA-binding domains. One of these, the zinc finger protein Odd-skipped, requires its eh1-like sequence for repressing specific target genes in segmentation (Goldstein, 2005).

The eh1 Gro recruitment domain was originally defined as a heptapeptide motif that is conserved in members of the En family of homeodomain proteins and their vertebrate homologues. More recently, eh1-dependent binding to Gro has also been demonstrated in vitro for various other Drosophila and mammalian proteins, nearly all of which contain homeodomains. Given that Bowl and Odd, two non-homeodomain ZnF transcription factors, contain this motif and interact with Gro, the possibility was explored that eh1 motifs are prevalent among additional non-homeodomain transcription factor families. Indeed, an unbiased yeast screen for Gro-interacting proteins selected two additional transcriptional regulators that contain eh1-like motifs, namely, Sloppy-paired (Slp; Forkhead related) and Dorsocross (Doc; T box). Alignment of the eh1-like sequences of Bowl, Odd, Slp, and Doc with those of En and Gsc revealed three conserved amino acids: phenylalanine-x-isoleucine-x-x-isoleucine (Phe-x-Ile-x-x-Ile, where x is any amino acid). Subsequent database searches for presumptive Drosophila transcription factors containing this minimal peptide sequence identified a wide range of potential negative regulators belonging to different superfamilies as classified by their distinct DNA-binding domain types. Remarkably, eh1-related motifs have been preserved in many human homologues of these fly proteins, indicating that the ability to bind Gro/TLE has been evolutionarily conserved in human transcriptional regulators and that this sequence may have been widely adopted throughout the proteome as a Gro recruitment domain (Goldstein, 2005).

Several representatives, corresponding to different transcription factor families, were tested for the ability to bind Gro in biochemical assays. Where possible, full-length expressed sequence tags encoding these proteins were obtained; otherwise, single exons containing the eh1-like sequence were PCR amplified from genomic DNA. Each polypeptide was assessed for the ability to pull down radiolabeled Gro in vitro. GST-tagged Slp and Doc (amino acids 254 to 391) readily retain Gro, as do Eyes absent (Eya) and the homeodomain proteins Ventral nervous system defective (Vnd, 1 to 465), Bagpipe (Bap, 1 to 129), BarH1, and Empty spiracles (Ems, 1 to 360), as well as the orphan nuclear hormone receptor DHR96. To confirm that these interactions rely on intact eh1-related sequences, the eh1 motif of one of these, BarH1, was mutated by substituting glutamic acid for Phe at position 1, finding that its binding to Gro is reduced by >60% (Goldstein, 2005).

DEVELOPMENTAL BIOLOGY

Embryonic

Northern analysis with gene-specific probes shows that all three Doc genes display similar expression profiles during development with maximal levels occurring between 2 and 12 hours of embryonic development and lower levels during late embryonic, larval and pupal stages. The only significant difference among the three genes in this assay is the expression of Doc1 mRNA in adult males: this was not observed for Doc2 and Doc3 (Reim, 2003).

The spatial expression patterns of Doc products in embryos were examined by whole-mount in situ hybridization with gene-specific probes and whole-mount immunocytochemistry using antibodies raised against the unique C-terminal regions of the Doc proteins. Since all three genes have essentially identical expression patterns (with some minor differences regarding the relative levels of expression in different tissues), they will be collectively referred as 'Doc genes'. As expected, Doc proteins are exclusively nuclear during interphase (Reim, 2003).

The initial expression of Doc genes is observed at the cellular blastoderm stage in a transverse stripe encompassing the dorsal ~40% of the embryonic circumference within the prospective head region. A short time later, a narrow longitudinal stripe of expression appears, which ultimately extends all along the dorsal midline of the embryo, and the joint domains form a cross-shaped pattern of Doc expression in dorsal areas of the early embryo. The domain of the transverse stripe is located anterior to the cephalic furrow, formed during gastrulation and largely corresponds to procephalic neuroectoderm. The cells of this domain continue Doc expression until stage 11, when the segregation of procephalic neuroblasts is completed. By contrast, the cells from the dorsal longitudinal domain within the trunk region give rise to amnioserosa, which maintains strong Doc expression until stage 15. In addition, the cells from the anterior and posterior termini within this longitudinal stripe contribute to regions of the anterior and posterior digestive tract and maintain expression until stage 11 (Reim, 2003).

During stages 9 and 10, a new pattern of Doc expression emerges within dorsolateral areas of the germ band from parasegment (PS) 1 to PS13; the pattern consists of 13 rectangular cell clusters. This metameric expression includes ectodermal as well as underlying mesodermal cells. In the ectoderm, Doc expression is excluded from the dorsalmost cells near the amnioserosa at this stage, whereas in the ectoderm the metameric Doc expression extends to the dorsalmost areas of this germ layer. To determine the segmental register of Doc expression in the mesoderm, embryos were co-stained with antibodies against the POU domain transcription factor Cf1a (Ventral veins lacking/Drifter), which mark the tracheal placodes. Doc and Cf1a are expressed in mutually exclusive domains, which implies that Doc expression encompasses prospective tissues of the lateral epidermis and dorsolateral sensory organs. After stage 11, the segmental expression in the epidermis is modified to form segmental stripes that are interrupted in dorsolateral regions. Within these stripes, Doc expression is largely found in posterior areas of the anterior compartments of each segment and there is a graded distribution of Doc expression with increasing levels towards the posterior of each stripe. The dorsal epidermal expression domains now extend to the amnioserosa, and after dorsal closure the bilateral domains merge at the dorsal midline (Reim, 2003).

Additional sites of Doc expression during late embryogenesis include the dorsal pouch in the embryonic head, the anterior pair of Malpighian tubules and the pentascolopidial chordotonal sensory organs. The mesodermal expression of the Doc genes is observed in areas between the expression domains of the homeobox gene bagpipe (bap) at stage 10. This location defines them as dorsal areas of the mesodermal A (or slp) domains, which include the dorsal somatic and cardiogenic mesoderm. During early stage 11, additional Doc expression initiates in the caudal visceral mesoderm, which contains the founder cells of the longitudinal muscles of the midgut. Two out of six bilateral cardioblasts in each segment of the dorsal vessel (Lo, 2001), that are tin negative and svp positive, also express the Doc genes (Reim, 2003).

The Dorsocross T-box genes are key components of the regulatory network controlling early cardiogenesis in Drosophila

Cardiac induction in Drosophila relies on combinatorial Dpp and Wg signaling activities that are derived from the ectoderm. Although some of the actions of Dpp during this process have been clarified, the exact roles of Wg, particularly with respect to myocardial cell specification, have not been well defined. The present study identifies the Dorsocross T-box genes as key mediators of combined Dpp and Wg signals during this process. The Dorsocross genes are induced within the segmental areas of the dorsal mesoderm that receive intersecting Dpp and Wg inputs. Dorsocross activity is required for the formation of all myocardial and pericardial cell types, with the exception of the Eve-positive pericardial cells. In an early step, the Dorsocross genes act in parallel with tinman to activate the expression of pannier, a cardiogenic gene encoding a Gata factor. Loss- and gain-of-function studies, as well as the observed genetic interactions among Dorsocross, tinman and pannier, suggest that co-expression of these three genes in the cardiac mesoderm, which also involves cross-regulation, plays a major role in the specification of cardiac progenitors. After cardioblast specification, the Dorsocross genes are re-expressed in a segmental subset of cardioblasts, which in the heart region develop into inflow valves (ostia). The integration of this new information with previous findings has allowed drawing a more complete pathway of regulatory events during cardiac induction and differentiation in Drosophila (Reim, 2005b).

In vertebrate species, genetic studies with loss-of-function alleles have implicated Tbx1, Tbx2, Tbx5 and Tbx20 in the control of heart morphogenesis and the regulation of cardiac differentiation markers. In the case of Tbx5, a small number of cardiac differentiation genes have been identified as direct downstream targets. However, owing to the complexity of the system, the respective positions of these genes within a regulatory network during early cardiogenesis are still poorly understood (Reim, 2005b).

Drosophila offers a simpler system to study regulatory networks in cardiogenesis. The Tbx20-related T-box genes mid and H15 have been shown to play a role in cardiac development downstream of the early function of the NK homeobox gene tin and the Gata gene pannier (pnr). Whereas the role of these genes in the morphogenesis of the cardiac tube is minor, they are involved in processes of cardiac patterning and differentiation during the second half of cardiogenesis, which includes the activation of tin expression in myocardial cells (Reim, 2005a). The present report characterizes the roles of the Tbx6-related Dorsocross T-box genes (which may actually have arisen from a common ancestor of the vertebrate Tbx4, Tbx5 and Tbx6 genes), in Drosophila cardiogenesis. The Doc genes have a fundamental early role; they are required for the specification of all cardiac progenitors that generate pure myocardial and pericardial lineages. They are not required for generating dorsal somatic muscle progenitors and lineages with mixed pericardial/somatic muscle, even though their early expression domains also include cells giving rise to these lineages (Reim, 2005b).

The new information on the regulation and function of Doc fills a major gap in the understanding of early Drosophila cardiogenesis. Previous data have shown that the combinatorial activities of Wg and Dpp are required for the formation of both myocardial and pericardial cells. In addition, the homeobox gene even-skipped (eve) is a direct target of the combined Wg and Dpp signaling inputs in specific pericardial cell/dorsal somatic muscle progenitors. Current data identify the Doc genes as downstream mediators and potential direct targets of combined Wg and Dpp signals during the induction of myocardial and Eve-negative pericardial cell progenitors. The induction of Doc expression by Wg and Dpp occurs concurrently with the induction of tin by Dpp alone, at a time when the mesoderm still consists of a single layer of cells. As a result, tin and Doc are co-expressed in a segmental subset of dorsal mesodermal cells that include the presumptive cardiogenic mesoderm. Conversely, in the intervening subset of dorsal mesodermal cells (the presumptive visceral mesoderm precursors) tin is co-expressed with bagpipe (bap) and biniou (bin), which are both negatively regulated by Wg via the Wg target sloppy paired (slp). Ultimately, these shared responses to Dpp, differential responses to Wg and the specific genetic activities of Doc versus bap and bin lead to the reciprocal arrangement of cardiac versus visceral mesoderm precursors in the dorsal mesoderm (Reim, 2005b and references therein).

Although the Dpp signaling pathway (and likewise, the Wg pathway) is activated in both ectodermal and mesodermal germ layers, tin and bap respond to it only in the mesoderm. The germ layer-specific response of these genes to Dpp relies on two probably interconnected mechanisms. The first of these involves the additional requirement for Tin protein as a mesodermal competence factor for Dpp signals, which is initially produced in the mesoderm downstream of twist. The second involves the specific repression of the responses of tin and bap to Dpp in the ectoderm by yet unidentified factors that bind to the Dpp-responsive enhancers of these two genes. By contrast, the Doc genes are induced by Dpp and Wg with the same spatial and temporal expression patterns in both germ layers. This implies that the (yet unknown) Dpp and Wg-responsive enhancer(s) of the Doc genes are not subject to the ectodermal repressor activities acting on the tin and bap enhancers, and fits with the observation that induction of Doc in the mesoderm does not require Tin as a mesodermal competence factor. However, because of the distinct roles of Doc in the ectoderm and mesoderm, this situation also implies that Doc must act in combination with germ layer-specific co-factors to exert its respective functions. These data suggest that, in the early mesoderm, Doc acts in combination with tin (Reim, 2005b).

A key gene requiring combinatorial Doc and Tin activities for its activation in the cardiac mesoderm is the Gata factor-encoding gene pannier (pnr). pnr expression is activated in the cardiac mesoderm shortly after the induction of Doc and tin, at a time when Doc expression has narrowed to the mesodermal precursors giving rise to pure cardiac lineages. The mechanisms restricting Doc expression to the cardiac mesoderm are currently not known, but as a consequence, pnr expression is also limited to the cardiac mesoderm. It is conceivable that Doc receives continued inputs during this period from the ectoderm through Dpp, whose expression domain narrows towards the dorsal leading edge by then. Together with the observed feedback regulation of pnr on tin and Doc, this situation leads to a prolonged co-expression of Tin, Doc and Pnr in the cardiac mesoderm of stage 11 to stage 12 embryos. Based upon the onset of the expression of early markers such as mid and svp, this is precisely the period when cardiac progenitors become specified (Reim, 2005b).

It is anticipated that the activation of some downstream targets in presumptive cardiac progenitors requires the combination of two, or perhaps all three, of these cardiogenic factors. Potential target genes include mid, svp and hand. However, none of these candidates is essential for generating cardiac progenitors, although mid and svp are known to be required for the normal diversification of cardioblasts within each segment (Reim, 2005b).

The observation that forced expression of Pnr in the absence of any Doc partially rescues cardiogenesis could indicate that the early, combinatorial functions of tin and Doc are primarily mediated by pnr. Alternatively, or in addition, this observation and the fact that a few cardioblasts can be generated without Doc could point to the existence of some degree of functional redundancy among these three factors. In the context of the latter possibility, it is tempting to speculate that the functional redundancy among T-box, Nkx and Gata factors during early cardiogenesis has further increased during the evolution of the vertebrate lineages. This would explain the less dramatic effects of the functional ablation of Tbx5, Nkx2-5 and Gata4/5/6 on vertebrate heart development as compared to the severe effects of Doc, tin or pnr mutations on dorsal vessel formation in Drosophila. Like the related Drosophila genes, these vertebrate genes are co-expressed in the cardiogenic region and developing heart of vertebrate embryos, which at least for Nkx2.5 and Gata6 also involves cross-regulatory interactions that reinforce their mutual expression (Reim, 2005b).

The observed co-expression of Doc, Tin and Pnr allows for the possibility that, in addition to combinatorial binding to target enhancers, protein interactions among these factors play a role in providing synergistic activities during cardiac specification. Physical interactions of Tbx5 with Gata4 and Nkx2-5, as well between Nkx2-5 and Gata4 in vitro as well as synergistic activities cell culture assays have been demonstrated in mammalian systems and may be relevant to human heart disease. In Drosophila, the genetic interactions between Doc, tin and pnr observed both in loss- and gain-of-function experiments reveal similar synergistic activities of the encoded factors during early cardiogenesis. Altogether, these observations make it likely that these Drosophila factors also act through combinatorial DNA binding and mutual protein interactions to turn on target genes required for the specification of cardiac progenitors (Reim, 2005b).

Whereas pnr is expressed only transiently during early cardiogenesis, tin and Doc continue to be expressed in developing myocardial cells, suggesting that they act both in specification and differentiation events. Recently it was shown that the T-box gene mid is required for re-activating tin in cardioblasts (Reim, 2005a). Of note, owing to the action of svp, Doc and tin are expressed in complementary subsets of cardioblasts within each segment. This mutually exclusive expression of tin and Doc implies that they are not acting combinatorially but, instead, act differentially during later stages of myocardial development. Hence, their activities could result in the differential activation of some differentiation genes such as Sulfonylurea receptor (Sur), which is specifically expressed in the four Tin-positive cardioblasts in each hemisegment (Nasonkin, 1999; Lo, 2001), and wingless (wg), which is only turned on in the two Doc-positive cells in each hemisegment of the heart that generate the ostia. Surprisingly, even the activation of some genes that are expressed uniformly in all cardioblasts has turned out to result from differential regulation within the Tin-positive versus Doc-positive cardioblasts. For example, regulatory sequences from the Mef2 gene for the two types of cardioblasts are separable and those active within the four Tin-positive cells are directly targeted by Tin. Likewise, regulatory sequences from a cardioblast-specific enhancer of Toll have been shown to receive differential inputs from Doc and Tin, respectively, in the two types of cardioblasts. In parallel with this differential regulation, it is anticipated that yet unknown differentiation genes are activated uniformly in all cardioblasts downstream of mid/H15 and hand. The integration of the new information on the roles of Doc in cardiogenesis has now provided a basic framework of signaling and gene interactions through all stages of embryonic heart development, which in the future can be further refined upon the identification of new components and additional molecular interactions (Reim, 2005b).

Larval

Dorsocross1 (Doc1) and Doc2/CG5187 are T-Box related factors that are expressed in what appears to be an identical domain in the wing disc. Both transcripts also accumulate in body wall cells and this probably lowers their position in the overall ranked list (Butler, 2003).

EFFECTS OF MUTATION

The similarities in sequence and expression of the three Doc genes suggests functional redundancy among these genes. Because molecular analysis of available deficiencies at 66E-F shows that none of them uncovered all three genes, the flanking P-insertions EP(3)3556 and EP(3)584 were used in attempts to delete the entire Doc gene cluster via male recombination-induced mutagenesis. Molecular mapping of the obtained deletions demonstrates that two of them, Df(3L)DocA and Df(3L)DocB, which were generated with the distally located insertion EP(3)3556 and caused embryonic lethality, delete all three Doc genes. Since Df(3L)DocA deletes the smallest number of additional genes (CG5087, CG5194, CG5144, Argk and CG4911), the phenotypic analysis in the present study using this deficiency is described, although the salient phenotypes are very similar between Df(3L)DocA and Df(3L)DocB (Reim, 2003).

Additional genetic analysis shows that it is possible to obtain a small number of viable adult escapers with the genotype Df(3L)Scf-R11/Df(3L)DocA, which indicates that CG5087 is not absolutely required for viability, and that Doc1 and Doc2 can functionally substitute for the loss of Doc3. Similarly, the full viability of flies with the genotype Df(3L)DocA/Df(3L)EP584MR2 shows that CG4911 and the 5' exons of Argk (preceding the large intron) are also not essential. Furthermore, it was determined that embryos with the genotypes Df(3L)Scf-R11/Df(3L)DocA and Df(3L)DocA/Df(3L)29A6 (which causes pupal lethality) do not display any of the phenotypes described below for Df(3L)DocA homozygous embryos. In summary, genetic analysis shows that the loss of either Doc3 or Doc1 can be compensated for by the remaining two Doc genes in embryos and that the phenotypes are a consequence of the loss of all three of the Doc genes. However, a contribution of CG5194, which encodes a 128 amino acid predicted ORF with no known homology, to the observed phenotypes, cannot be ruled out (Reim, 2003).

Because of the prominent Doc expression in the primordia and developing tissue of the amnioserosa, the amnioserosa marker Krüppel (Kr) was used to examine whether the Doc genes are required for the development of this extra-embryonic tissue. These experiments demonstrate that homozygous Df(3L)DocA mutant embryos (henceforth called DocA mutants) fail to express Kr in the amnioserosa at any stage, whereas CNS expression of Kr is not affected. To confirm that this observed phenotype is due to the loss of Doc gene function, Doc gene functions were reduced by using RNA interference (RNAi) as an independent assay and rescue experiments with DocA mutants were performed. Injection of a mixture of equimolar amounts of dsRNAs for all three Doc genes frequently results in a complete absence of Kr expression in the amnioserosa. The remaining embryos display strongly reduced numbers of Kr-containing nuclei in this tissue. These phenotypes correlate with the observed absence or severe reduction of Doc protein levels in Doc RNAi embryos. Hence, the strongest phenotype obtained by RNAi mimics the observed DocA mutant phenotype, confirming that the lack of Kr expression in DocA mutant embryos is specifically due to the loss of the activity of all three Doc genes (Reim, 2003).

Besides the effects on Kr expression, DocA mutant and RNAi-treated embryos share several morphological defects. The extending germ band is unable to displace the amnioserosa fully towards the anterior and the posterior germ band is therefore forced to bend underneath the amnioserosa. Of note, germ band retraction is strongly disrupted, which can be clearly seen in stage 14 embryos and in cuticle preparations of unhatched first instar larvae. This phenotype is shared with previously described genes of the u-shaped group, which affect the maintenance of the amnioserosa. Kr expression and the germ band retraction defects in DocA mutant embryos can be partially rescued by expressing any of the three Doc genes with an early amnioserosa-specific driver. Rescue with Doc2 is consistently more efficient when compared with Doc1 and Doc3, although it is not known whether this difference is due to a higher intrinsic activity or a more efficient expression of Doc2 protein in this assay (Reim, 2003).

An additional phenotype consists of reductions in the size of the embryonic head in DocA mutants and RNAi-treated embryos, which is apparent from stage 12 onwards and results in reduced head structures and a frequent failure of head involution at later stages. This phenotype is probably due to excessive cell death as a consequence of the absence of Doc activity in the procephalic neuroectoderm and other dorsal areas of the embryonic head. The observed head phenotypes, as well as the aberrant shape of the filzkörper, are also reminiscent of similar phenotypes of embryos mutant for genes of the ush group (Reim, 2003).

To obtain more information about the particular role of the Doc genes in the specification and/or differentiation of the amnioserosa, the distribution of additional amnioserosa markers was analyzed in DocA mutant embryos. For the ush group gene hnt a strong reduction of expression is found, with significant levels of Hnt protein being detected in nuclei only along the posterior margin of the amnioserosa. By contrast, the expression of the amnioserosa marker race (Ance) (is initiated normally in the primordium of the amnioserosa of DocA mutant embryos, suggesting that the expression of the race upstream activator zerknüllt (zen) is also not disrupted. However, after embryonic stage 9, race expression is gradually lost in the amnioserosa of DocA mutant embryos and its residual mRNA distribution closely follows that of Hnt (Reim, 2003).

The expression of a novel amnioserosa marker, which is encoded by the homeobox gene C15, was examined. In the normal situation, C15 is expressed in the amnioserosa from stage 7 until stage 17, when the amnioserosa undergoes apoptosis. In addition, from early stage 10 onwards there is a narrow domain of expression at the leading edge of the dorsal germ band, which later becomes segmental. In DocA mutant embryos, the level of C15 expression in early amnioserosa cells is unaltered; this allows use of C15 protein as a marker for the development of this tissue in the absence of Doc activity (Reim, 2003).

Until stage 9, the large majority of amnioserosa nuclei in DocA mutant embryos appear large and flattened as in wild-type embryos. Together with data from alpha-tubulin staining, this observation indicates that the amnioserosa cells begin to acquire the normal features of a squamous epithelium. However, the amnioserosa does not display a properly folded morphology during stages 8-10, and the posterior germ band is forced to bend towards the inside in DocA mutant embryos. In addition, some small nuclei become detectable within the amnioserosa during this stage. Altogether, these observations indicate that the amnioserosa initiates its differentiation process in the absence of Doc gene activity but fails to complete it, thus leading to morphological and functional abnormalities of this tissue towards the end of germ band elongation. Much stronger alterations can be observed during subsequent stages, when there are an increasing number of C15-stained amnioserosa nuclei with much smaller diameters than regular amnioserosa nuclei. At late stage 12, almost all amnioserosa cells feature small nuclei that are difficult to distinguish from dorsal epidermal cells. Co-staining for race indicates that it is predominantly the cells with the small nuclei that lose race expression, while most normally-sized nuclei are still surrounded by race signals. From this stage onwards, non-stained 'holes' appear in the amnioserosa and the number of C15-stained amnioserosa nuclei decreases prematurely. Hence, unlike wild-type embryos, stage 14 DocA mutant embryos are not covered dorsally by C15-stained amnioserosa cells. In addition to the observed alterations in the amnioserosa, the C15 expression domain at the leading edge of the epidermis appears significantly broadened (Reim, 2003).

Is the increasing number of smaller nuclei in the amnioserosa of DocA mutant embryos connected with abnormal cell divisions? The M-phase marker phospho-Histone H3 can be detected in numerous amnioserosa nuclei of DocA mutant embryos after stage 10; this increase is not seen in wild-type embryos. In addition, there is significant incorporation of BrdU in amnioserosa nuclei of DocA mutant embryos (particularly in the small nuclei, whereas no incorporation is observed in wild-type embryos. Mitotic spindles are also present in the amnioserosa of DocA mutants. These observations indicate that the normal G2 arrest of amnioserosa cells has been released and the cells re-enter the cell cycle. Whether the subsequent disappearance of small C15-stained amnioserosa nuclei in DocA mutant embryos is a result of premature apoptosis of cells in this tissue was also tested. This possibility was confirmed by the results of TUNEL labeling experiments, which produced signals in many amnioserosa nuclei from 12 onwards. Most of the TUNEL-labeled nuclei have reduced or are lacking C15 expression, which shows that wild-type amnioserosa nuclei at late stage 12 are not apoptotic). Altogether, these observations suggest that loss of Doc activity prevents the normal differentiation of the amnioserosa to a fully functional tissue, suspends the cell cycle block of amnioserosa cells, and causes premature apoptotic cell death in this tissue (Reim, 2003).

The Drosophila homolog of vertebrate Islet1 is a key component in early cardiogenesis

In mouse, the LIM-homeodomain transcription factor Islet1 (Isl1) has been shown to demarcate a separate cardiac cell population that is essential for the formation of the right ventricle and the outflow tract of the heart. Whether Isl1 plays a crucial role in the early regulatory network of transcription factors that establishes a cardiac fate in mesodermal cells has not been fully resolved. This study analyzed the role of the Drosophila homolog of Isl1, tailup (tup), in cardiac specification and formation of the dorsal vessel. The early expression of Tup in the cardiac mesoderm suggests that Tup functions in cardiac specification. Indeed, tup mutants are characterized by a reduction of the essential early cardiac transcription factors Tin, Pnr and Dorsocross1-3 (Doc). Conversely, Tup expression depends on each of these cardiac factors, as well as on the early inductive signals Dpp and Wg. Genetic interactions show that tup cooperates with tin, pnr and Doc in heart cell specification. Germ layer-specific loss-of-function and rescue experiments reveal that Tup also functions in the ectoderm to regulate cardiogenesis and implicate the involvement of different LIM-domain-interacting proteins in the mesoderm and ectoderm. Gain-of-function analyses for tup and pnr suggest that a proper balance of these factors is also required for the specification of Eve-expressing pericardial cells. Since tup is required for proper cardiogenesis in an invertebrate organism, it is appropriate to include tup/Isl1 in the core set of ancestral cardiac transcription factors that govern a cardiac fate (Mann, 2009).

The specification of a subset of mesodermal cells towards a cardiac fate requires well-orchestrated interactions of a plethora of factors. Drosophila is the model system of choice to decipher the complex transcriptional network that initiates and sustains a cardiac lineage. The data place tup as an essential component in the early transcriptional network that specifies cardiac mesoderm (Mann, 2009).

After the initially broad expression domain of Tin has become restricted to the dorsal mesodermal margin, Tup expression is first seen in the cardiac mesoderm in ~10 small clusters, which co-express Eve. Slightly later, Tup is present throughout the Tin-positive cardiac mesoderm and gene expression analyses in tup^isl-1, tin³⁴⁶, pnr^VX6 and Df(3L)DocA embryos demonstrate that all four factors are required to maintain each other's expression. Additionally, analyses of cardiac gene expression in embryos that are transheterozygotic for tup and tin, pnr or Doc, showed that these factors interact genetically to specify heart cells (Mann, 2009).

Although it might be expected that Tup expression is lost in tin mutants since these embryos are devoid of heart cells, it is interesting that Tup expression in the early cell clusters is still initiated. This finding is somewhat reminiscent of the observation that the initiation of Doc expression is also independent of tin. According to the temporal appearance of Tup in the cardiac mesoderm with respect to Tin and Doc, tup is required for their maintenance rather than their initiation. By contrast, the onset of mesodermal Pnr and Tup expression appears to coincide. It was not resolved whether Tup is induced by Pnr or directly by Dpp. A direct regulation by Dpp was implicated by the reduced expression of Tup after mesodermal overexpression of UAS-brinker, which is known to bind to dpp-response elements of dpp target genes. Conversely, it was shown that dpp expression depends on tup and the present data suggest that this regulation requires pnr (Mann, 2009).

Germ layer-specific inhibition of Tup using a construct that lacks the homeodomain, but contains the two LIM domains, revealed that Tup can regulate cardiogenesis in the mesoderm as well as from the ectoderm. Since the 69B-Gal4 driver has been reported not to be strictly ectodermal, it is possible that mesodermal Tup function was also interfered with. However, the mesodermal expression of 69B-Gal4 seems to be negligible. The effect of ectodermal Tup inhibition on cardiogenesis in the mesoderm can only be explained if the function of a secreted growth factor is impaired. dpp expression was analyzed, and a slight downregulation of its transcripts was observed in embryos expressing UAS-tupδHD in the ectoderm. Since this effect might not be sufficient to account for the strong Tin phenotype, further experiments will be required to determine whether additional growth factors are affected (Mann, 2009).

To better determine the germ layer-specific contribution of Tup in cardiogenesis, attempts were made to rescue the Tin phenotype by co-expressing the full-length tup cDNA. Somewhat unexpectedly, a better rescue was obtained when both constructs were expressed in the ectoderm rather than in the mesoderm. Since the LIM domains present in tupδHD can sequester LIM-domain-binding proteins, a simple explanation for this finding is that Tup interacts with proteins that are present in the mesoderm but not in the ectoderm. It is reasonable to hypothesize that in the mesoderm the LIM domains of tupδHD not only act as a dominant-negative for Tup, but additionally for another, perhaps as yet unidentified, LIM-domain containing protein. Since it has been shown that Pnr can bind Tup through the LIM domains, it is likely that Pnr function was interfered with by overexpressing UAS-tupδHD. The requirement of the LIM domains for proper cardiac specification is shown by the reduction of Tin-expressing cells after mesodermal expression of the UAS-tupδLIM construct. Further experiments are under way to better resolve the molecular function of Tup in the different tissues (Mann, 2009).

Since the mesodermal expression of UAS-tupδHD resulted in a strong reduction of Tin-expressing cells at early stages of cardiac mesoderm formation, it was surprising to observe a rather low reduction of Dmef2-positive myocardial cells at later stages (15/16). To exclude the possibility that the twi-Gal4 driver does not sufficiently express UAS-tupδHD throughout embryogenesis, this experiment was repeated using the combined mesodermal driver twi-Gal4; 24B-Gal4. However, the phenotypes were not enhanced. A time course for Tin expression in these crosses revealed that Tin appears to recover over time. A similar phenomenon can be seen in tup^isl-1 mutants, although it might not be as obvious because the mutants also lack ectodermal tup expression. In any case, the data is suggestive of a different temporal requirement for tup with respect to tin expression. It is known that tin expression depends on different transcriptional activation events. Consistent with the onset of Tup expression in the cardiac mesoderm at mid-stage 11, the earlier phases of Tin expression are unlikely to depend on Tup. Hence, the initial Tin expression at stages 8-10 is sufficient to generate a considerable number of Dmef2-positive myocardial cells at later stages (Mann, 2009).

These analyses further implicate that Tup might act as a transcriptional activator or repressor depending on the cellular context and on the factors with which it is co-expressed. This is most strikingly observed with respect to the Odd-expressing pericardial and lymph gland cells. In tup mutants, Odd-positive cells are missing in both organs. A similar phenotype is seen when Tup is overexpressed in the mesoderm using the twi-Gal4 driver. The loss of Odd-expressing cells in lymph glands is reminiscent of the phenotype observed in tup mutants, although it is less severe. This differential occurrence of the phenotype indicates that tup can differentially regulate factors involved in cardiogenesis versus lymph gland development. This is substantiated by the finding that mesodermal overexpression of tup results in an increase in Hand expression in the lymph glands, while Hand expression throughout the dorsal vessel is only mildly affected. Despite the loss of Odd-positive cells after early mesodermal tup overexpression, Tup is required in the pericardial and lymph gland cells at later stages to maintain Odd expression. Moreover, overexpressing tup in the pericardial cell lineage yields additional Odd-expressing pericardial cells and rescues Odd expression in the lymph glands (Mann, 2009).

To obtain more insight into possible functional interactions with other cardiac transcription factors, tup was overexpressed in combination with pnr^D4. The latter is a highly active variant of wild-type pnr that contains an amino acid substitution in the N-terminal zinc finger, which abolishes binding of Ush to Pnr. Mesodermal overexpression of pnr^D4 results in robust ectopic activation of Tin and embryos co-overexpressing tup and pnr^D4 exhibit the same phenotype. Most likely, a possible influence of Tup on Pnr activity, regardless of whether it is positive or negative, is concealed by the strong gain-of-function pnr allele. However, analysis of Eve expression does provide insight into possible regulatory interactions between Tup and Pnr. Mesodermal overexpression of each factor alone yields opposing phenotypes, and when both factors are co-overexpressed Pnr^D4 can efficiently counteract Tup activity and prevent the overspecification of Eve cells. Vice versa, Tup can, although only moderately, counteract the effect of Pnr^D4. It has been shown that during patterning of the thorax, Tup can antagonize the proneural activity of Pnr by forming a heterodimer, and that the physical interaction between Pnr and Tup is mediated by the two zinc fingers of Pnr. Hence, the somewhat weak, but possibly antagonistic, function of Tup towards Pnr^D4 in Eve-positive cell specification could be due to the amino acid substitution encoded in the pnr^D4 allele, which might weaken the interaction between the two factors, as compared with wild-type Pnr. Overexpression of a Tup construct that lacks both LIM domains did not result in expanded Eve-positive clusters, which strongly suggests that the effect of Pnr on Tup activity, as seen when both factors are co-expressed, requires the presence of the LIM domains (Mann, 2009).

In summary, these data demonstrate the crucial role of tup in the proper specification of cardiac mesoderm in an invertebrate organism. Therefore, tup/Isl1 should be added to the core set of ancestral cardiac transcription factors. Consequently, this implicates that the evolution of the vertebrate four-chambered heart does not necessarily require the acquisition of a novel network of cardiac transcription factors. At least, it is unlikely that tup/Isl1 is part of a regulatory network separate from that of tin/Nkx2.5, pnr/Gata4 and Doc/Tbx5/6 because it is an essential factor for the formation of the simple linear heart tube in the fly (Mann, 2009).

Spire, an actin nucleation factor, regulates cell division during Drosophila heart development

The Drosophila dorsal vessel is a beneficial model system for studying the regulation of early heart development. Spire (Spir), an actin-nucleation factor, regulates actin dynamics in many developmental processes, such as cell shape determination, intracellular transport, and locomotion. Through protein expression pattern analysis, this study demonstrates that the absence of spir function affects cell division in Myocyte enhancer factor 2-, Tinman (Tin)-, Even-skipped- and Seven up (Svp)-positive heart cells. In addition, genetic interaction analysis shows that spir functionally interacts with Dorsocross, tin, and pannier to properly specify the cardiac fate. Furthermore, through visualization of double heterozygous embryos, it was determined that spir cooperates with CycA for heart cell specification and division. Finally, when comparing the spir mutant phenotype with that of a CycA mutant, the results suggest that most Svp-positive progenitors in spir mutant embryos cannot undergo full cell division at cell cycle 15, and that Tin-positive progenitors are arrested at cell cycle 16 as double-nucleated cells. It is concluded that Spir plays a crucial role in controlling dorsal vessel formation and has a function in cell division during heart tube morphogenesis (Xu, 2012).

Proper dorsal vessel morphogenesis is critically dependent upon intercellular signaling and the regulation of gene expression. Although great progress has been made in the study of heart development, it is not known exactly how many genes and pathways are involved in this cardiogenic process or how many of these factors cooperate together. Previous genetic screens have identified genes that play roles in the specification, morphogenesis, and differentiation of the heart, including mastermind and tup. The current sensitized screen has also proved to be an efficient method to find additional factors in this process, suggesting that much remains to be learned about the molecular components involved in correct dorsal vessel formation (Xu, 2012).

Spir is required for the proper timing of cytoplasmic streaming in Drosophila, and loss of spir leads to premature microtubule-dependent fast cytoplasmic streaming during oogenesis, the loss of oocyte polarity, and female sterility. Even though it is known that spir is an important actin filament nucleation factor, the findings are the first report to describe a function of spir for cell division. Through phenotypic analysis of the spir mutant phenotype, it was found that many cardioblast nuclei are partially or completely divided. However, the cytoplasm is not divided in the absence of spir, which is consistent with the function of spir in cytoplasmic movement. Thirteen rapid nuclear division cycles without cell division initiate Drosophila embryo development, followed by three waves of cell division. The first wave of cell division occurs in the mesoderm at cell cycle 14. After this initial division, cells migrate, spread dorsally and undergo a second round of cell division at cell cycle 15. The third wave of cell division in the mesoderm occurs at the end of germband extension during cell cycle 16. There are two different types of cardioblast precursor cells: one type divides into two identical Tin-positive cardioblasts (TC), and the other type divides into one Svp-positive cardioblast (SC) and one Svp-positive pericardial cell (SPC). Based on the comparison of CycA and spir mutant phenotypes, a tentative cell division model is proposed to demonstrate spir function in determining cardiac cell fate (see A cell division model of spir function during heart development). In a wild-type background, one Svp-positive super progenitor (SSP) divides into two Svp-positive progenitors (SP), then each of these cells divides into one SPC and one SC. For Tin-positive super progenitors (TSP), after each divides into two Tin-positive progenitors (TP), each TP further divides into two identical TCs. In the current model, division from the super progenitor to progenitors takes place at cell cycle 15, and division from progenitors to full differentiated heart cells occurs at cell cycle 16. In CycA mutants, mitosis 16 is blocked such that both SPs and TPs stop cell division. This results in the two SPs assuming a myocardial fate. Thus the number of SCs remains normal, but half of the TCs are missing in the CycA mutants. The data suggest that in spir mutant embryos, most of the SPs fail to undergo full cell division at cycle 15 resulting in a SPC fate with paired nuclei. A subset of these cells are able to undergo the 15th cell division but are arrested at cycle 16 as double-nucleated cells which exhibit both Svp and Mef2 staining, characteristic of the SCs seen in the CycA mutants. Similarly, for TPs, cycle 16 was also blocked such that it resulted in two double-nucleated cells. In summary, Spir affects mitosis 16 for Tin-positive cell division and both mitosis 15 and 16 for Svp-positive cell division (Xu, 2012).

Antibody staining suggests that Spir is expressed ubiquitously before stages 12-13 and is located in both nuclei and cytoplasm. After cell cycle 16 cell division stops, occurring during stage 10-11. The expression of Spir in the cytoplasm then decreases gradually. At stage 15, the staining pattern shows mostly nucleus expression with some cytoplasmic expression and by stage 16 the nuclei become distinct indicating nucleus staining only. It is hypothesized that expression of Spir decreases in the cytoplasm but remains constant in the nuclei when cell division halts (Xu, 2012).

The genetic analysis of spir, Doc, pnr and tin suggests that these factors may regulate each other during dorsal vessel formation, and especially significant is the interaction between spir and pnr. Pnr is a GATA class transcription factor expressed in both the dorsal ectoderm and dorsal mesoderm, where it is required for cardiac cell specification. Proper dorsal vessel formation is inhibited in pnr loss-of-function embryos due to failure in the specification of the cardiac progenitors. In spir mutants, the expression pattern of Pnr remains normal. However, Spir is over-expressed in the cardiac mesoderm in pnr mutants, suggesting that Pnr may repress the expression of the spir (Xu, 2012).

In conclusion, Spir is a newly-identified factor functioning in cell division during dorsal vessel formation. Tin-, Eve- and Svp-positive heart cells are all affected in the absence of spir. Also, spir expression depends on the transcription factors Doc, tin and pnr. Genetic interaction data also show that spir cooperates with CycA in heart cell division (Xu, 2012).

EVOLUTIONARY HOMOLOGS

Related mammalian genes include human Tbx6 and murine Tbx2. For information about Doc homologs see Optomotor blind: Evolutionary homologs

REFERENCES

Search PubMed for articles about Drosophila Dorsocross1, Dorsocross2 and Dorsocross3

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date revised: 2 December 2022

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