InteractiveFly: GeneBrief

H15 and Midline: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene names - H15 and midline

Synonyms - neuromancer (nmr)

Cytological map position - 25E5

Function - T-box transcription factor

Keywords - heart, segment polarity

Symbols - H15 and mid

FlyBase IDs: FBgn0016660 and FBgn0261963

Genetic map position - 2-16

Classification - T-box transcription factors

Cellular location - nuclear

H15 NCBI links: Precomputed BLAST | Entrez Gene

Midline NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Svendsen, P. C., Ryu, J. R. and Brook, W. J. (2015). The expression of the T-box selector gene midline in the leg imaginal disc is controlled by both transcriptional regulation and cell lineage. Biol Open [Epub ahead of print]. PubMed ID: 26581591
The Drosophila Tbx20 homologs midline and H15 act as selector genes for ventral fate in Drosophila legs. midline and H15 expression defines the ventral domain of the leg and the two genes are necessary and sufficient for the development of ventral fate. Ventral-specific expression of midline and H15 is activated by Wingless (Wg) and repressed by Decapentaplegic (Dpp). This study identifed VLE, a 5 kb enhancer that drives ventral specific expression in the leg disc that is very similar to midline expression. Subdivision of VLE identifies two regions that mediate both activation and repression and third region that only mediates repression. Loss- and gain-of-function genetic mosaic analysis shows that the activating and repressing regions respond to Wg and Dpp signaling respectively. All three repression regions depend on the activity of Mothers-against-decapentaplegic, a Drosophila r-Smad that mediates Dpp signaling, and respond to ectopic expression of the Dpp target genes optomoter-blind and Dorsocross 3. However, only one repression region is responsive to loss of schnurri, a co-repressor required for direct repression by Dpp-signaling. Thus, Dpp signaling restricts midline expression through both direct repression and through the activation of downstream repressors. midline and H15 expression are both subject to cross-repression and feedback inhibition. Finally, a lineage analysis indicates that ventral midline-expressing cells and dorsal omb-expressing cells do not mix during development. Together this data indicates that the ventral-specific expression of midline results from both transcriptional regulation and from a lack of cell-mixing between dorsal and ventral cells.
Fu, C. L., Wang, X. F., Cheng, Q., Wang, D., Hirose, S. and Liu, Q. X. (2016). The T-box transcription factor Midline regulates wing development by repressing wingless and hedgehog in Drosophila. Sci Rep 6: 27981. PubMed ID: 27301278
Wingless (Wg) and Hedgehog (Hh) signaling pathways are key players in animal development. However, regulation of the expression of wg and hh are not well understood. This study shows that Midline (Mid), an evolutionarily conserved transcription factor, expresses in the wing disc of Drosophila and plays a vital role in wing development. Loss or knock down of mid in the wing disc induced hyper-expression of wingless (wg) and yielded cocked and non-flat wings. Over-expression of mid in the wing disc markedly repressed the expression of wg, DE-Cadherin (DE-Cad) and armadillo (arm), and resulted in a small and blistered wing. In addition, a reduction in the dose of mid enhanced phenotypes of a gain-of-function mutant of hedgehog (hh). Repression of hh was observed upon overexpression of mid in the wing disc. Taken together, it is proposed that Mid regulates wing development by repressing wg and hh in Drosophila.

The Drosophila melanogaster genes midline and H15 encode predicted T-box transcription factors homologous to vertebrate Tbx20 genes. All identified vertebrate Tbx20 genes are expressed in the embryonic heart and both midline and H15 are expressed in the cardioblasts of the dorsal vessel, the insect organ equivalent to the vertebrate heart. The midline mRNA is first detected in dorsal mesoderm at embryonic stage 12 in the two progenitors per hemisegment that will divide to give rise to all six cardioblasts. Expression of H15 mRNA in the dorsal mesoderm is detected first in four to six cells per hemisegment at stage 13. The expression of midline and H15 in the dorsal vessel is dependent on Wingless signaling and the transcription factors tinman and pannier. The selection of two midline-expressing cells from a pool of competent progenitors is dependent on Notch signaling. Embryos deleted for both midline and H15 have defects in the alignment of the cardioblasts and associated pericardial cells. Embryos null for midline have weaker and less penetrant phenotypes while embryos deficient for H15 have morphologically normal hearts, suggesting that the two genes are partially redundant in heart development. Despite the dorsal vessel defects, embryos mutant for both midline and H15 have normal numbers of cardioblasts, suggesting that cardiac cell fate specification is not disrupted. However, ectopic expression of midline in the dorsal mesoderm can lead to dramatic increases in the expression of cardiac markers, suggesting that midline and H15 participate in cardiac fate specification and may normally act redundantly with other cardiogenic factors. Conservation of Tbx20 expression and function in cardiac development lends further support for a common ancestral origin of the insect dorsal vessel and the vertebrate heart (Miskolczi-McCallum, 2005).

A second study by Qian (2005) provides more detailed information on a cellular fate switch accompanying loss- and gain-of-function of this gene pair. Referring to midline and H15 by the alternative name neuromancer (nmr) the Qian study shows that gene function causes a switch in cell fates in the cardiogenic region, in that the progenitors expressing the homeobox gene even skipped (eve) are expanded, accompanied by a corresponding reduction of the progenitors expressing the homeobox gene ladybird (lbe). As a result, the number of differentiating myocardial cells is severely reduced whereas pericardial cell populations are expanded. Conversely, pan-mesodermal expression of nmr represses eve, while causing an expansion of cardiac lbe expression, as well as ectopic mesodermal expression of the homeobox gene tinman. In addition, nmr mutants with less severe penetrance exhibit cell alignment defects of the myocardium at the dorsal midline, suggesting nmr is also required for cell polarity acquisition of the heart tube. In exploring the regulation of nmr, it was found that the GATA factor Pannier is essential for cardiac expression, and acts synergistically with Tinman in promoting nmr expression. Moreover, reducing nmr function in the absence of pannier further aggravates the deficit in cardiac mesoderm specification. Taken together, the data suggest that nmr acts both in concert with and subsequent to pannier and tinman in cardiac specification and differentiation. It is proposed that nmr is another determinant of cardiogenesis, along with tinman and pannier (Qian, 2005).

The insect heart, or dorsal vessel, is a simple muscular tube that pumps hemolymph and hematocytes through the open circulatory system. Heart development in vertebrates and dorsal vessel development have many features in common. During gastrulation, the trunk mesoderm of the fly invaginates along the ventral midline. The mesoderm flattens, forming a single cell layer inside the ectoderm, and migrates dorsally. The fly heart forms from bilateral rows of cells at the leading edge of the migrating mesoderm. The rows fuse along the dorsal midline forming a linear tube. A similar process initiates vertebrate heart development, where bilateral fields of mesoderm migrate and fuse to form a linear heart tube prior to the looping and subsequent morphogenesis that forms a multichambered heart. In addition to these morphological similarities, signaling from other germ layers is required for the cardiac potential of the mesoderm tube and a conserved transcription factor network specifies cardiac fate in both Drosophila and vertebrates (Miskolczi-McCallum, 2005).

The Drosophila NK-2 class homeodomain transcription factor Tinman is required for the specification of the dorsal mesoderm [heart development is completely abolished in tinman mutants]. After mesoderm involution, tin is initially expressed in all mesoderm cells under the control of the bHLH factor Twist. During stages 9 and 10, tin becomes restricted to a broad domain in the dorsal trunk mesoderm through maintenance by signaling from overlying ectoderm, which secretes the BMP family protein Decapentaplegic (Dpp). By mid stage 11, tin expression is maintained in segmental clusters of dorsal mesoderm cells by ectoderm and mesoderm secreting the Wnt protein Wingless (Wg). These tin-expressing domains contain the precursors for the heart including progenitors of both the contractile cardioblasts and the noncontractile pericardial cells. The dorsal vessel is not specified in the absence of tin function or if Dpp or Wg signal transduction is blocked (Miskolczi-McCallum, 2005 and references therein).

Tin regulates cardiac gene expression in conjunction with the GATA factor Pannier (Pnr). In stage 11, pnr is expressed in the mesoderm under the control of ectodermal Dpp signaling and mesodermal Tin. Null mutations in pnr greatly diminish heart development, and ectopic pnr expression is sufficient to induce excess cardiac fate. Expression of pnr in the mesoderm is also required to maintain proper tin expression. Ectopic expression of tin has little effect on heart formation, but in combination with pnr, ectopic tin has a synergistic effect on the ability of mesoderm to adopt cardiac fate. This synergy is likely to occur at the level of the regulatory sequences of cardiac genes as Tin and Pnr form complexes in vitro and heart-specific enhancers require clusters of Tin and Pnr binding sites (Miskolczi-McCallum, 2005 and references therein).

NK-2 homeodomain and GATA transcription factors are also critical for heart development in vertebrates. Loss of function for Nkx2.5, the vertebrate tin homologue, causes arrested heart development at the linear tube stage. Furthermore, Nkx2.5 activates cardiac gene expression in a complex with GATA4. Genetic analysis of the roles of GATA factors in vertebrate heart development is complicated by potential genetic redundancy of GATA4, 5, and 6, but several lines of evidence suggest that GATA factors are also required to establish cardiac fate (Miskolczi-McCallum, 2005).

More recently, T-box transcription factors have been identified as regulators of vertebrate heart development that interact with both Nkx2.5 and GATA proteins. Tbx5 (Drosophila homolog: Optomotor-blind), the gene responsible for Holt-Oram syndrome, is required for chamber formation. Mutations in Tbx5, Nkx2.5, and GATA4 all cause similar heart defects in humans and Tbx5 interacts physically with both Nkx2.5 and GATA4. Mutations in GATA4 that abrogate the Tbx5-GATA4 physical interaction in vitro cause heart defects similar to those seen in Tbx5 mutants, indicating that the physical interactions are functionally important. All pairwise combinations of Tbx5, Nkx2.5, and GATA4 activate gene expression synergistically in vitro. The cardiac expression of another T-box family member, Tbx20 (also called Tbx12), is conserved in all vertebrates (Ahn, 2000; Carson, 2000; Griffin, 2000; Iio, 2001; Kraus, 2001; Meins, 2000). Zebrafish embryos exhibit heart defects following injection with morpholino oligonucleotides directed against H15-related-T-box (hrT), a zebrafish Tbx20 homolog (Szeto, 2002). Tbx20 shares some of the properties of Tbx5 in vitro, forming complexes with Nkx2.5 and GATA4 (Plageman, 2004; Stennard, 2003) and synergistically activating cardiac gene expression in some assays (Stennard, 2003). However, the relationship between Tbx20 and Tbx5 may be complex: Tbx20 acts to inhibit Tbx5 transcription in vivo (Szeto, 2002; Takeuchi, 2003), and in some assays (Plageman, 2004) can block transcriptional activation by Tbx5 in vitro (Miskolczi-McCallum, 2005).

Clearly, T-box proteins are key regulators of heart development in vertebrates, which raises the question of whether T-box function is conserved in Drosophila dorsal vessel development. T-box genes have duplicated and diverged throughout metazoan evolution, acting in several developmental processes in a wide variety of organisms. In the human and mouse genomes, there are 18 identified T-box genes representing five subfamilies, while the fly genome contains eight T-box genes representing four of the five subfamilies found in mice and humans. There is no clear Tbx5 homolog in Drosophila, and the Drosophila gene most similar to Tbx5, optomotor-blind (omb), is not expressed in the dorsal vessel. Dorsocross 1, 2, and 3 (Doc1, 2, and 3), the Tbx6 homologs, are expressed in a subset of cardioblasts. The Doc genes play a key role in the subdivision of the dorsal mesoderm, but it has not been shown if they play a role specifying cardiac fate and the expression of Tbx6 is not conserved in vertebrate hearts. Therefore, the best candidates for Drosophila cardiogenic T-box genes are the Tbx20 homologs H15 (Brook, 1996) and midline (mid) (Buescher, 2004). H15-lacZ, a lacZ enhancer trap inserted adjacent in the H15 mid locus, is expressed in all cardioblasts (Griffin, 2000), the contractile myocardial cells of the dorsal vessel (Miskolczi-McCallum, 2005).

In order to assess whether the requirement for T-box transcription factors is conserved in heart and dorsal vessel development, the expression, regulation, and function of mid and H15 in the development of the Drosophila dorsal vessel were examined. The expression of the genes was shown to correlate with the specification of cardioblast fate -- the genes are required in a partially redundant fashion in heart morphogenesis, and ectopic expression of mid is sufficient to cause dorsal mesoderm cells to acquire cardiac fate. Based on these results, it is argued that Tbx20 genes represent another component of the conserved transcription factor network acting in heart and dorsal vessel development (Miskolczi-McCallum, 2005).

Therefore, the Tbx20 homologs mid and H15 are expressed in cardioblasts and are required in a partially redundant manner for dorsal vessel morphogenesis. Furthermore, the activation of mid in the dorsal mesoderm depends on cardiogenic factors and mid expression correlates with cardioblast specification. Finally, ectopic expression of mid induces cardiac fate in the dorsal mesoderm in a manner similar to the effects of ectopic tin and pnr expression. Together, these results suggest an important role for mid and H15 in the specification and differentiation of cardiac fate. Vertebrate T-box transcription factors are critical regulators of heart development. Tbx20 is essential for morphogenesis of the vertebrate heart and interacts with the Tin and Pnr homologs Nkx2.5 and GATA4 in gene regulation (Plageman, 2004; Stennard, 2003; Szeto, 2002). Thus, the previously reported conservation of a cardiac regulatory network, including the NK-2 homeodomain proteins and the GATA factors, may now be extended to include T-box transcription factors. These findings offer further support for the proposal that, despite differing greatly in morphology, the vertebrate heart and the insect dorsal vessel may share a common evolutionary ancestor (Miskolczi-McCallum, 2005).

The timing and pattern of mid expression suggest that it is an early specific marker of cardioblasts. mid expression in the dorsal mesoderm is first observed in two cells per hemisegment in early stage 12, increasing to six cardioblasts per hemisegment throughout stages 12 and 13. Analysis of the cardioblast lineage with genetic mosaics and cell cycle manipulation indicates that the six cardioblasts in each hemisegment must derive from two progenitor cells. Furthermore, Notch signaling is required to restrict the number of cardioblasts in the dorsal vessel, and it was found that the number of cells expressing mid in the dorsal mesoderm is also restricted by Notch activation. Thus, these data are consistent with a model where mid-expressing cardioblast progenitors are normally selected from a cluster of competent cells by a signal or signals acting in opposition to Notch. The nature of this signal is unknown, although it is noted that the two mid-expressing cells arise in consistent positions relative to the ectodermal Wg and Engrailed stripes of cells and thus may respond to spatial cues emanating from the overlying ectoderm or arising from within the mesoderm (Miskolczi-McCallum, 2005).

Both mid and H15 are expressed in all cardioblasts from stage 13 onwards and the dorsal vessel is disorganized in H15 mid null embryos. Analysis of mid and H15 mutant embryos suggests that mid has the greater effect, causing some heart defects on its own while H15 is nonessential. However, loss of both genes has a stronger phenotype, suggesting that H15 augments the activity of mid. A similar relative requirement is found for the two genes in the segmentation of the embryonic ectoderm (Buescher, 2004). Normal numbers of cardioblasts, expressing markers of cardiac (tin) and muscle (DMef2) fate specification, as well as muscle differentiation (Myosin Heavy Chain), are formed in H15 mid null embryos. The cardioblasts migrate dorsally but do not always align properly with their contralateral partners. The pericardial cells are often displaced laterally or medially, and the lymph gland is enlarged due to increased cell number. Given these defects and the restriction of mid and H15 expression to cardioblasts, the essential function of mid and H15 may be to regulate the later stages of cardioblast morphogenesis (Miskolczi-McCallum, 2005).

Understanding the essential function of mid and H15 in dorsal vessel development awaits identifying the genes regulated by the two T-box transcription factors. Cardioblasts require specific cell polarity, adhesion, and migration in order to make the proper contacts with other cardioblasts and pericardial cells to form the tube of the dorsal vessel. Many cell adhesion molecules are expressed in cardioblasts and mutations in these genes cause dorsal vessel defects that are similar to those caused by loss of mid and H15. The misalignment of pericardial cells may be a secondary consequence of the defects in cardioblast morphogenesis. Similarly, the increase in lymph gland cell number is likely to be a nonautonomous effect since mid and H15 are not expressed in the lymph gland primordia. Because this defect results from an increase in the lymph gland cell number, it is possible that H15 mid mutant embryos are defective in a signal from the dorsal vessel regulating cell proliferation in the lymph gland (Miskolczi-McCallum, 2005).

The specification of cardiac fate in Drosophila is complex. No single factor has been identified that is both necessary and sufficient to induce mesoderm to form heart cells. Combined Dpp and Wg signaling are essential for heart development and can induce ectopic heart fate, but only in tin-expressing cells. Tin is essential for the specification of dorsal mesoderm and directly regulates many genes expressed in cardioblasts and pericardial cells. However, ectopic expression of tin has little effect on cardiac fate. By comparison, Pnr, a transcriptional cofactor of Tin, is able to induce extensive ectopic cardiac fate. However, some cardioblasts still form in pnr null embryos, albeit in greatly reduced numbers, indicating that pnr is not absolutely essential for heart cells to form. A formally similar situation is found for mid in that it is also sufficient but not necessary (even in the absence of H15) for cardiac fate. Recently, it has been shown that the Xenopus Tbx20 homolog interacts physically with GATA4 and Nkx2.5 (Plageman, 2003; Stennard, 2003). Furthermore, Tbx20 can have a synergistic effect on the activation of cardiac promoters in vitro with Nkx2.5 and GATA4 (Stennard, 2003). Thus, it is possible that Mid and H15 act as cofactors for Tin and Pnr in the regulation of cardiac fate specification in the fly. The expression of mid in the dorsal mesoderm is much more restricted than that of either tin or pnr, thus ectopic expression of mid may promote ectopic cardiac fate through interaction with Tin and Pnr. This interpretation is supported by the restriction of ectopic cardiac fate induced by mid to the dorsal mesoderm (Miskolczi-McCallum, 2005).

An interaction with Tin and Pnr may explain why mid induces ectopic cardioblasts, but it does not explain why mid and H15, or pnr for that matter, are not essential for cardioblast specification. A simple possibility is that another transcription factor may compensate for the absence of mid and H15. This could be either an unrelated factor or another T-box protein, for example, the Doc1, 2, and 3 Tbx6-like proteins, which are restricted to the svp-expressing cardioblasts. Assessing mid and H15 for redundancy with the Doc genes, not a trivial undertaking, because loss of function for Doc blocks embryonic development prior to heart morphogenesis, will be necessary to test this model. Another mechanism that may help compensate for the absence of mid and H15 is redundancy with Tin and Pnr activity. Given the pairwise synergy of GATA4, Nkx2.5, and Tbx20 in transcriptional assays (Stennard, 2003), it may be that removal of any one trans-acting factor would be insufficient to completely block cardiac fate. Either Pnr or Mid could be sufficient to induce some cardiac fate as long as Tin or other factors are present. In this context, it is worth noting that removal of tin, which has the greatest effect, completely blocking cardioblast fate specification, also prevents the expression of pnr, mid, and H15. In contrast, tin, mid and H15 are only partially reduced in pnr mutants, and tin and pnr are widely expressed in the dorsal mesoderm prior to the activation of mid and H15 expression. Analysis of genetic and molecular interactions between tin, pnr, mid, and H15 will be necessary to test these models (Miskolczi-McCallum, 2005).

The Midline protein regulates axon guidance by blocking the reiteration of neuroblast rows within the Drosophila ventral nerve cord

Guiding axon growth cones towards their targets is a fundamental process that occurs in a developing nervous system. Several major signaling systems are involved in axon-guidance, and disruption of these systems causes axon-guidance defects. However, the specific role of the environment in which axons navigate in regulating axon-guidance has not been examined in detail. In Drosophila, the ventral nerve cord is divided into segments, and half-segments and the precursor neuroblasts are formed in rows and columns in individual half-segments. The row-wise expression of segment-polarity genes within the neuroectoderm provides the initial row-wise identity to neuroblasts. This study shows that in embryos mutant for the gene midline, which encodes a T-box DNA binding protein, row-2 neuroblasts and their neuroectoderm adopt a row-5 identity. This reiteration of row-5 ultimately creates a non-permissive zone or a barrier, which prevents the extension of interneuronal longitudinal tracts along their normal anterior-posterior path. While the nature of the barrier is not know, the axon tracts either stall when they reach this region or project across the midline or towards the periphery along this zone. Previously studies have shown that midline ensures ancestry-dependent fate specification in a neuronal lineage. These results provide the molecular basis for the axon guidance defects in midline mutants and the significance of proper specification of the environment to axon-guidance. These results also reveal the importance of segmental polarity in guiding axons from one segment to the next, and a link between establishment of broad segmental identity and axon guidance (Manavalan, 2013).

Guiding axon growth cones towards their synaptic targets is one of the most fundamental processes during neurogenesis. Axon growth cones navigate through different regions by responding to cues from the environment to ultimately find their synaptic targets. While the two major signaling pathways, Slit-Robo and Net-Fra, provide a larger architecture for axon guidance within the nerve cord, local environment is expected to influence axon guidance as well. However, this particular aspect has not been examined in detail. A given local environment will be determined by the identity of the neuroectoderm, neurons, ectoderm and perhaps by the identity of the mesoderm as well. Thus, broad changes in local environment in which axon growth cones have to navigate is likely alter the route or guidance of these axons (Manavalan, 2013).

It is well established that segment polarity genes determine the broad identity of cells within the nerve cord just as they do later during development to determine the segmental identity within the epidermis. Segmentation genes, specifically the segment polarity genes such as Wg, Ptc, Hh, En are expressed in rows of NBs to define specific and row-wise NB identities. That these segmentation genes also play a role in axon guidance is indicated by the fact that mutations in many segmentation genes alter axon guidance. However, given that these mutations also alter NB identity, the effect of mis-specification of neuronal identity versus broad changes in the environment in which axons navigate, on axon guidance has been experimentally difficult to separate (Manavalan, 2013).

The work described in this paper, however, attempts to separate the role of identity versus environment and reveal the significance of local environmental niche to normal axon guidance. The results show that in mid mutants, there is an ectopic expression of segment polarity genes such as Wg, Gsb, Slp (and perhaps many more) in row 2 cells within the developing nerve cord, thus, re-specifying this row of cells into more like row 5 cells (and a second parasegmental boundary). This re-specification appears to ultimately create a zone or a barrier that prevents axon growth cones from progressing further in their normal route. Instead, such growth cones either stall or project peripherally or across the midline but along this zone of non-permissive barrier. The highly specific nature of the phenotype(s) in response to a specific change in the environment in mid mutants presents a classic example of the specificity of the environment to axon guidance. The results also show that the identity of some of the pioneering neurons, whose axon projections are misrouted, is not affected by loss of function for mid (Manavalan, 2013).

It would have been ideal if it were possible to identify a single molecule that makes this re-specified row of cells non-permissive to longitudinal tracts extension in their usual path. It is not known if such a molecule exists, or the mechanism that created the barrier. But the barrier is unlikely due to the ectopic expression of genes such as Wg, instead it must be due to the change in the row-identity, activating a distinct genetic program that does not permit axonal extension in their normal path. Ectopic expression of Wg, or Slp or Gsb simply reflects this change. It is also pointed out that this re-specified row 5 cells may not have all the features/genetic programs of a bona fide row 5 cells and more likely have a mixed identity. This is suggested by the fact that Slp and Gsb or even Wg expression in the re-specified row is not exactly like in a bona fide row 5. Similarly, it is not known at what point in development this zone or barrier is put in place, but it indeed originates with the altered row identity, and certainly becomes active by the time of pathfinding. This barrier might be due to signals from other neurons generated by the transformed NB row, or the transformed neuroectoderm/ectoderm. It is further pointed out that a clonal analysis experiment would have been desirable to show that a broad identity-change is necessary for the observed guidance defects. However, mid is expressed extensively in the germline, both in the soma and the germ cells, and there is a requirement for Mid in these cells. Ultimately, it may require isolation of a temperature sensitive allele in mid to address this question in an unequivocal way (Manavalan, 2013).

The results show that Mid does not regulate the expression of slit, robo or fra genes in the CNS. Consistent with this, the axon guidance defects in mid are distinct from the defects in slit or robo mutants. This was confirmed by several different ways: immunostaining, RNA whole mount in situ, Western analysis, qPCR and genetic interaction studies. A previous study (Liu, 2009) has suggested that Mid regulates sli, robo and fra. That was based the conclusion on finding a strong transheterozygous genetic interaction between mid and sli, and mid and fra, detected using BP102 staining of embryos that are transheterozygous for mid1 and sliGA20 and mid1 and fra3. Furthermore, it was reported that levels of fra mRNA and Fra and Robo proteins in mid mutant embryos were down regulated, and that this can be completely rescued by expressing mid using elev-GAL4 driver. It was also reported that ectopic expression of mid in salivary glands induces expression of robo and slit. Most of these previously reported effects were not observed in the current study. For example, no genetic transheterozygous interactions were observed between mid and sli mutants. Transheterozygous interactions are rare given the negative evolutionary impact of such interactions to survival, but when observed, it is usually with mutations in receptor-ligand pairs, or with gain of function/neomorphic situations). This study used stronger allelic combinations than the ones used by Liu with mid and sli. For mid, not only mid1 was used, but also a deficiency that removes both mid and its sister gene H15, as well as los1 (a midline allele). For slit, sli2, which is the strongest loss of function allele and genetically behaves as a null, was used. Furthermore, no such transheterozygous interactions were observed between mid and fra (Manavalan, 2013).

Secondly, it was found that while the ectopic expression of mid in salivary glands induced robo expression as was reported by Liu, no such induction was observed with slit. One should also consider the fact that Mid, Robo (and Slit) have mostly non-overlapping domains of expression in the CNS, therefore, the direct regulation of robo by Mid in the salivary gland has little relevance in the CNS or CNS development mediated by Robo or Mid. The results indeed bears this out. Not only the axon guidance defects are different between mid and slit or robo, the transcription of robo, slit or fra are also unaffected in mid mutants. There was some reduction in the levels of Slit in los1 allele in the midline in the PC region. But, the molecular lesion in los1 is complex and might have some allele-specific gain of function effects that alters cellular identity or function of the corresponding midline glia to mediate reduction in the Slit level in this region. Since no such reduction in the levels are seen in other mid alleles and more importantly the transcription of slit is unaffected in the deficiency that removes mid (and H15), it is thought that the slight reduction in the levels of Slit in los1 is allele-specific (Manavalan, 2013).

In the case of robo, the promoter has three TBEs. With three sites, Mid is more likely to be able to induce robo in an ectopic site. However, within the CNS, no significant loss of Robo expression was found in mid mutant embryos by immunohistochemistry (either in los1 or mid H15 deficiency embryos) or by Western analysis or robo transcription by qPCR. A slight reduction in the levels of Robo seen in Westerns is likely due to a secondary effect of loss of tracts and perhaps loss of some of the Robo-expressing neurons perhaps due to identity changes. The reason for the significant reduction in the expression of Robo in mid observed by Liu is not clear. It is thought that this may be due to some technical reasons such as variability from embryo to embryo to fixing and staining. Because of this possibility, this study followed a simple rule: in this case, focus was placed on mid mutant embryos that had strong guidance defects to determine if such mutant embryos also had a strong or weak expression of Robo and Slit. It was found that embryos with strong guidance defects also had strong Robo (or Slit) expression. Thus, selecting sub-stained mutant embryos and comparing them to optimally stained wild type embryos was avoided. Finally, Liu reported that there is overlapping expression of Mid and Slit in a small number of cells located laterally within the nerve cord. It is possible that Mid regulates slit expression in these cells, however, the contribution of Slit or such a regulation of slit to the overall axon guidance mediated by Slit is not clear and likely very minimal, if there is any. Whether mid affects netrin gene expression was not examined (Manavalan, 2013).

The current work shows that in mid mutants, the majority of axon growth cones of the longitudinal tracts stall and club together at the level of AC, creating a blob of axons, thus leading to interruptions between neuromeres. Interestingly, some of the tracts project outward towards the periphery or inward across the midline. This outward projection route is quite revealing: the projection path is mostly perpendicular to the midline and just below the transformed row of cells. The transformed row of cells corresponds to the region right above the AC or where the tracts stall. The most consistent change is seen with row 2 cells, changing into row 5 cells. How does these changes relate to wild type? In wild type, row 5 cells normally separate one neuromere from the next and also define PSB. Thus, the change from row 2 to row 5 in mid must be creating an environment that either lacks the necessary permissive/attractive cues or possess cues that are inhibitory to the projection of these axon tracts, causing the tracts to stall (Manavalan, 2013).

For example, in wild type, row 5 cells are located between pCC and vMP2, the two axons that pioneer the Fas II-positive medial tract. The growth cone from vMP2 in wild type only marginally encounters row 5 cells but does not necessarily traverse row 5. This is due to the fact that vMP2 is located in row 5 and the growth cone from a vMP2 stops at row 5 region and fasciculates with the vMP2 of the next hemisegment. However, it does encounter row 2 cells midway through the projection path. In mid mutants, since row 2 cells change to row 5, creating a region that vMP2 growth cone is perhaps normally programmed to stop. For the proper guidance of medial tract, normal projection of vMP2 and pCC is necessary and loss of either of the two pioneer neurons causes aberrant medial tract guidance. Therefore, it seems likely that vMP2 stalls and the pCC projection, along with several other follower projections, also stalls; or some of the tracts project across the midline or away towards the periphery. In fact, these abnormal projection patterns, especially towards the periphery appear to be guided by the newly created barrier. This situation is also the same for MP1 or dMP2 (Manavalan, 2013).

What is the mechanism within the re-specified row of cells that eventually mediates the block for axon projection? The re-specified rows of cells would have a whole set of new (row 5-specific) genetic programs that may simply not conducive to longitudinal tracts. Additionally, the role of Ephrin pathway in axon guidance may be relevant here. The Drosophila Ephrin (Eph), which is a transmembrane protein, is shown to prevent interneuronal axons from exiting the Drosophila embryonic CNS; some of the interneuronal pathways in mid mutant exit the nerve cord. Ephrin/Eph signaling is via cell-to-cell contact and depends on the clustering of Eph receptors and their ligands. This multimerization activates the kinase activity of the receptor and leads to the phosphorylation of the receptor within the cytoplasm-exposed tail region and the binding of downstream effectors. This triggers the depolymerization of actin in growth cones, modifying the Integrin-based cell adhesion. The CNS-exiting phenotype of interneuronal pathways in mid mutants suggests a possible de-regulation of the Eph-pathway. But, it may also be that changes in Eph or similar cell-adhesion mechanism mediate the formation of the barrier and exiting of some of the interneuronal pathways from the CNS (Manavalan, 2013).

Previous results show that Mid acts as a transcriptional repressor of gsb-n. However, in mid mutants the transformation of row 2 into row 5 also activates Gsb expression. The ectopic activation of Gsb in these cells in mid, however, is not a direct de-repression of gsb, but an indirect consequence of the transformation of cell identity from row 2 (a Gsb-negative row of cells) to row 5, a Gsb-positive row. Finally, the results provide clear evidence that segmentation genes can regulate axon guidance via broadly defining cellular identity, creating a permissive and non-permissive boundaries or niche. It is also emphasized that extrapolating expression relationships to functional relevance from induction in ectopic sites, in vitro and tissue culture experiments, bioinformatics or other similar in vitro studies carry inherent risks and should be done with caution (Manavalan, 2013).


Transcriptional Regulation

In order to determine where mid and H15 fit in the genetic hierarchy controlling heart development, their expression was examined in several mutant backgrounds. The initiation of mid expression in the dorsal mesoderm in early stage 12 occurs after the expression of tin and pnr, as well as after the period of Wg signaling in the dorsal mesoderm, suggesting that mid and H15 are regulated downstream of the factors that confer cardiac fate. Indeed, the dorsal vessel expression of mid and H15 is completely lost in both wgcx4 and tinec40 mutant embryos, which fail to specify dorsal mesoderm. Embryos mutant for pnr have greatly decreased numbers of cardioblasts. Accordingly, mid and H15 expression is variably lost in pnrvx6 null mutant embryos, with most embryos completely lacking mid expression in the dorsal mesoderm. Ectopic expression of pnr throughout the mesoderm using the GAL4/UAS system is able to induce ectopic expression of mid and H15. These results indicate that the initiation of mid and H15 in the dorsal mesoderm is downstream of factors required for the specification of cardiac fate (Miskolczi-McCallum, 2005).

Drosophila T box proteins break the symmetry of hedgehog-dependent activation of wingless

Segmentation of the Drosophila embryo is a classic paradigm for pattern formation during development. The Wnt-1 homolog Wingless (Wg) is a key player in the establishment of a segmentally reiterated pattern of cell type specification. The intrasegmental polarity of this pattern depends on the precise positioning of the Wg signaling source anterior to the Engrailed (En)/Hedgehog (Hh) domain. Proper polarity of epidermal segments requires an asymmetric response to the bidirectional Hh signal: wg is activated in cells anterior to the Hh signaling source and is restricted from cells posterior to this signaling source. This study reports that Midline (Mid) and H15, two highly related T box proteins representing the orthologs of zebrafish hrT and mouse Tbx20, are novel negative regulators of wg transcription and act to break the symmetry of Hh signaling. Loss of mid and H15 results in the symmetric outcome of Hh signaling: the establishment of wg domains anterior and posterior to the signaling source predominantly, but not exclusively, in odd-numbered segments. Accordingly, loss of mid and H15 produces defects that mimic a wg gain-of-function phenotype. Misexpression of mid represses wg and produces a weak/moderate wg loss-of-function phenocopy. Furthermore, it has been shown that loss of mid and H15 results in an anterior expansion of the expression of serrate (ser) in every segment, representing a second instance of target gene repression downstream of Hh signaling in the establishment of segment polarity. The data presented indicate that mid and H15 are important components in pattern formation in the ventral epidermis. In odd-numbered abdominal segments, Mid/H15 activity plays an important role in restricting the expression of Wg to a single domain (Buescher, 2004).

The larval cuticle of Drosophila is a model system for generating patterns from fields of cells. The ventral cuticle exhibits a segmentally reiterated array of six rows of unique denticles separated by areas of naked cuticle. These external structures reflect the cellular diversity within the underlying epidermis, and defective cuticle patterns are indicative of incorrectly specified cell fates. The secreted products of two segment polarity genes, wg and hh, are the key players that initiate progressive patterning events that ultimately result in epidermal differentiation at the single-cell level. Thus, patterning requires the tight regulation of the spatial limits of Wg and Hh expression. In early embryogenesis, pair-rule gene activity initiates the expression of Wg and Hh in adjacent stripes, with Wg just anterior to the Hh-expressing cells. After stage 9, reciprocal signaling between Wg- and Hh-expressing cells stabilizes their expression domains. Acting anisotropically, Hh signaling activates Wg anterior, but not posterior, to the Hh stripe. Finally, Wg expression becomes independent of Hh and is maintained through an autoregulatory feedback loop. Previous studies have led to the conclusion that Hh signaling is bidirectional because it maintains patched (ptc)-gene expression in narrow stripes anterior and posterior to the En/Hh stripe. The ptc gene product is a repressor of Wg expression, and maintenance of Wg expression at stage 9 requires the Hh-mediated derepression. However, despite the symmetry of Hh signaling, the outcome with respect to Wg expression is asymmetric and results in a single Wg stripe anterior to the En/Hh stripe. To rationalize the differential response of Wg to the Hh signal, a model has been put forward that subdivides each parasegment into two domains: the posterior half of the parasegment represents the wg-competent domain, and the anterior half is the en-competent domain. The wg-competent domain encompasses those cells that express Wg in a ptc mutant background. Additional studies have shown that wg competence requires the activity of the pair-rule/segment polarity genes sloppy-paired 1, 2 (slp1, 2), which are expressed in broad stripes anterior to the En/Hh stripe. It has been suggested that Slp permits the activation of Wg anterior to the En/Hh stripe by antagonizing one (or several) putative repressor(s) of Wg (Buescher, 2004 and references therein).

This study reports that Mid and H15 act to repress the Hh-dependent activation of Wg in the en-competent domain predominantly in odd-numbered segments. Furthermore, these results suggest that the Slp-mediated repression of Mid/H15 anterior to the En/Hh stripe is an important component of wg competence (Buescher, 2004).

Mutant alleles of mid (mid1, mid2), recessive embryonic-lethal zygotic mutations, were first identified in the classic screen for segmentation genes (Nüsslein-Volhard, 1984). An additional allele, midGA174, was isolated from a collection of EMS-induced mutations. mid mutant larvae are characterized by patches of naked cuticle in the ventral-most part of the abdominal denticle belts. In addition, a near-complete loss of denticle belts (or segmental halves of denticle belts) was occasionally observed. Both aspects of the phenotype are more pronounced in odd-numbered segment, whereas even-numbered segments show similar but milder defects. Genetic mapping (Nüsslein-Volhard, 1984) has placed mid at cytological position 25E (Buescher, 2004).

This region was examined for genes with expression patterns that suggest a role in segmentation. A P element insertion upstream of H15 (CG6604) displays a ß-gal expression pattern consistent with such a role (Griffin, 2000). Both H15 and an adjacent gene, CG6634, encode highly homologous T box proteins with essentially identical expression patterns ; thus, both genes represented candidates for mid. Deletion of H15 by X-ray resulted in a homozygous pharate adult lethal line (H15x4) with no appreciable cuticle phenotype. In contrast, sequencing of CG6634 DNA from homozygous mid1, mid2, and midGA174 embryos reveals nonsense mutations. The putative translation products of mid1 and midGA174 lack the DNA binding domain and are most probably nonfunctional. These data indicate that CG6634 encodes the mid gene. This conclusion is corroborated by the observation that ectopic expression of CG6634 in the mid1 mutant background rescues the cuticle defect. To determine if, in the absence of Mid function, H15 contributes to cuticle formation, early larvae lacking both copies of H15 and one copy of mid (H15x4/Df-GpdhA) were examined. A weak mid phenotype was observed. Removal of both copies of H15 and mid (hereafter referred to as mid/H15) results in a strong enhancement of the phenotype; the denticle rows 1-5 are frequently lost in odd-numbered segments, whereas even-numbered segments show milder defects. These findings suggest that Mid and H15 act redundantly to control denticle formation (Buescher, 2004).

The mid RNA expression pattern is typical of segment polarity genes. H15 RNA expression pattern is identical with the exception that expression levels up to stage 9 are significantly lower than mid expression levels. Fourteen stripes of mid expression are first detected in stage 5 embryos. Alternating stripes differ in width and intensity; even-numbered stripes are wider and show higher levels of expression. (Note that the first five mid stripes localize to the presumptive head and thoracic segments. Therefore, the even-numbered stripes 6, 8, 10, and 12 correspond to the stripes that are found in the odd-numbered abdominal segments 1, 3, 5, and 7.) During germband extension, expression becomes more uniform in consecutive stripes and is approximately equal in all stripes from stage 8 onward. mid-expressing stripes are maintained until the end of embryogenesis. However, mid expression occurs in distinct phases, during which it is regulated by different factors. Dynamic changes of the mid stripes with respect to width and location reflect the different regulatory inputs (Buescher, 2004).

The precise location of the mid stripes was first determined by double labeling wild-type embryos with a mid RNA in situ probe and an anti-Wg antibody. During stages 5-11, mid expression abuts the posterior side of the Wg stripe, but from late stage 11 onward, the mid and the Wg stripes are separated by two rows of cells that express neither gene. The Wg and mid stripes remain separated until the end of embryogenesis. To determine the posterior limits of mid expression, wild-type embryos were stained with the mid RNA in situ probe and an anti-En antibody. Up to stage 9, mid expression is found in the En/Hh cells, but even-numbered mid stripes extend farther to include two rows of cells just posterior to the En/Hh domain. Weaker mid expression is found posterior to the En/Hh domain in odd-numbered segments. This early expression of mid depends on pair-rule gene activity. By stage 10, the mid-expressing domain contracts to coincide with the En/Hh stripe. This stripe persists until early stage 11, after which mid expression in the En domain decays. The maintenance of mid expression during stages 9-11 within the En domain requires Wg function; in wg null mutant embryos (wgCX4), mid expression decays prematurely from stage 9 onward and is absent at stage 11. This dependence on Wg may provide a rationale for the narrowing of the mid stripe after initiation by the pair-rule genes because Wg signaling is not effective posterior to the En domain. During late stage 11, mid expression is reinitiated in a 2- to 3-cell-wide stripe posterior to the En stripe. This stripe persists until the end of embryogenesis. This late expression of mid is sensitive to Hh signaling; raising the level of Hh signaling (enGal4; UAS-hh) results in a posterior expansion of the mid stripe. It is noteworthy that this late expression of mid represents another example of an asymmetric outcome of bidirectional Hh signaling (Buescher, 2004).

In odd-numbered abdominal segments, and to a lesser extent in even-numbered segments, concomitant loss of both Mid and H15 results in an excess of naked cuticle similar to that caused by ectopic Wg expression. To investigate if loss of Mid/H15 affects the Wg expression pattern, double-mutant embryos were stained with a anti-Wg antibody or a wg RNA in situ probe. From stage 9 onward, ectopic 1-cell-wide Wg stripes were observed in odd-numbered abdominal segments and weak patches of ectopic Wg were occasionally found in even-numbered segments. The initially weak ectopic stripes subsequently increased in intensity so that by stage 13 high levels of ectopic Wg were found in odd-numbered segments and lower levels of ectopic Wg were seen in some even-numbered segments. Hence, the pair-rule-biased pattern of ectopic Wg expression reflects the defect observed in mid/H15 larval cuticles; namely, this defect is a gain of naked cuticle predominantly, but not exclusively, in odd-numbered segments. Ectopic Wg expression was also found in stage 9 mid single mutants, albeit less frequently and less robustly. It was also observed very rarely in H15 single mutants. The precise location of the ectopic Wg expression was determined by double staining mid/H15 mutant embryos with anti-Wg and anti-En antibodies. The ectopic Wg stripes abut the posterior side of the En stripes (within the domain of early mid expression, generating a pattern in which En-expressing cells are straddled by Wg-expressing cells (Buescher, 2004).

To assess the effects of mid gain-of-function on Wg expression, several Gal4 drivers were used to misexpress mid. The cuticle phenotype of the resulting larvae was examined; this phenotype represents a highly sensitive read-out of even small changes in the level of Wg signaling. The cuticle phenotype of ptcGal4;UAS-mid and scaGal4;UAS-mid larvae mimicked that of a late loss of wg function: in the posterior half of the larvae, nearly all naked cuticle was replaced with denticles. In the anterior half, extra denticles appeared predominantly in the ventral-most region. This phenotype appeared with 100% penetrance. In addition, ptcGal4;UAS-mid larvae (and to a lesser extent scaGal4;UAS-mid embryos) showed specific morphological defects characteristic of wg loss-of-function mutants -- reduced body size, strong segmental indentations, and malformation of the head. To confirm that the cuticle defects caused by ectopic Mid were due to decreased Wg expression, UAS-mid, UAS-wg, or both were expressed under the control of prdGal4, which is strongly expressed in the Wg domain in the even-numbered abdominal segments. Coexpression of wg with mid resulted in near-complete suppression of the fusion of alternating denticle belts as a result of misexpression of mid alone. The cuticle defects observed in ptcGal4;UAS-mid larvae were matched by altered levels of Wg protein: from stage 10 onward, Wg protein in the ventral ectoderm decayed, and at stage 13 it was nearly absent in the ventral epidermis. Hence, misexpression of mid is sufficient to antagonize Wg in its endogenous expression domain (Buescher, 2004).

The appearance of the ectopic Wg stripes in mid/H15 mutants coincides with the stabilization phase of the endogenous Wg expression by Hh signaling. The endogenous Wg expression (anterior to the En/Hh stripe) was not affected in either mid single or mid/H15 double mutants at any time, suggesting that Hh signaling is normal. To corroborate this conclusion, the expression of the hh gene, the distribution of Hh protein, and the effect of Hh signaling were examined on the expression of the ptc gene. ptcRNA expression in narrow stripes anterior and posterior to the En domain has been shown to be a read-out of bidirectional Hh signaling. No difference was observed in the hh and ptc expression patterns in wild-type and mid/H15 embryos. This demonstrates that the regulatory interactions that result in the maintenance of endogenous Wg at stage 9 and the autoregulation of Wg from stage 11 onward function normally in mid/H15 double mutants. This raises the possibility that Hh signaling may activate Wg expression in a symmetrical manner but that expression of Wg posterior to the En stripe is antagonized by Mid/H15. To investigate this possibility, the effects were studied of manipulating Hh signaling in a mid/H15 mutant background. When the level of Hh signaling was raised from within the endogenous Hh/En domain (enGal4;UAS-hh), the ectopic Wg expanded to a 2- to 3-cell-wide stripe, thus demonstrating that the level of Hh controls the spatial limits of ectopic Wg expression. Reciprocally, in hh/mid/H15 triple mutants, no ectopic Wg expression was observed. These data show that Hh signals symmetrically with respect to cells anterior and posterior to the En/Hh stripe and Mid and H15 are required to prevent posterior Wg expression, thus ensuring that Wg expression remains restricted to a single domain (Buescher, 2004).

Loss of mid/H15 leads to ectopic Wg expression in a single row of cells. Raising the level of Hh signaling (mid/H15;enGal4;UAS-hh) results in an expansion of the ectopic Wg stripe. To unmask the potential of cells to express Wg in the absense of Mid-mediated repression, mid1ptc9 double-mutant embryos in which Wg expression is largely independent of Hh were examined. In ptc mutant embryos, the expression domain of Wg broadens in the anterior direction, and ectopic En expression is induced de novo in cells anterior to these broadened domains. The anterior region of the segment between the ectopic En stripe and the next endogenous En stripe does not express Wg. Double labeling of ptc embryos with the mid RNA in situ probe and anti-En antibody showed that mid fills this region. Removal of Mid function caused all cells in odd-numbered segments to express either Wg or En (but not both) and resulted in a complete loss of the odd-numbered denticle belts (Buescher, 2004).

Close examination revealed some unexpected features: the wg transcription domain is slightly widened in mid1ptc9 embryos as compared to ptc single mutants; however, this occurs in all segments without any pair-rule bias. Moreover, consecutive wg RNA stripes form pairs (1-2, 3-4, and so on) that are separated by wider gaps from the subsequent pair. The region between two partners of each wg pair is entirely filled with En-expressing cells, suggesting a fusion of the ectopic and the endogenous En domains. Taken together, these data indicate that concomitant loss of mid and ptc results in defects that go beyond a simple change of Wg and En expression. This notion is supported by the observation that, as compared to even-numbered segments, odd-numbered segments in mid1ptc9 embryos are shorter by approximately two rows of cells. Future studies will show if mid plays a role in proper segment formation, which requires cell survival, cell division, and cell sorting (Buescher, 2004).

Earlier studies of the regulation of Wg expression showed that loss of ptc results in an anterior expansion of the wg transcription domain. It does not, however, result in ectopic Wg expression posterior to the En domain. To rationalize this asymmetric response of the wg promoter to bi-directional Hh signaling, it was proposed that each parasegment is divided into two domains: the posterior half of the parasegment represents the wg-competent domain and the anterior half is the en-compentent domain. Wg-competence requires the slp genes which are expressed in broad stripes anterior to the En/Hh stripe. This study has shown that loss of mid/H15 results in the ectopic expression of Wg within the en-competent domain (where Slp is absent). This prompted a study to examined any possible regulatory interactions between slp and mid/H15. Double staining of H15-lacZ embryos (which are viable and wild-type with respect to Wg expression) with a slp in situ probe and anti-ß-gal antibody showed that, at stage 9, slp and H15 (mid) are expressed in non-overlapping domains that are separated by one row of cells in the center of each segment. Within this central row, slp-positive and mid-positive cells intermingle with cells expressing neither gene (Buescher, 2004).

It is conceivable that the ectopic expression of Wg in mid/H15 mutant embryos could be a secondary effect brought about by a gain of Slp expression posterior to the En stripe. RNA in situ analysis revealed no change of the Slp expression pattern in mid/H15 embryos. Hence, in odd-numbered segments of wild-type embryos, the lack of wg competence in the cells immediately posterior to the En/Hh stripe is a consequence of Wg repression via Mid/H15 rather than of a lack of activation via Slp (Buescher, 2004).

Previous work has suggested that Slp permits the Hh-dependent activation of Wg anterior to the En/Hh stripe by antagonizing a repressor of Wg. Based on the data presented above, Mid/H15 appear to be such repressors. To determine if Slp is a negative regulator of mid expression, the effect of slp loss-of-function and slp misexpression was studied on the distribution of mid RNA. In slp mutant embryos the early mid expression is normal. However from early stage 9 onward the mid stripes broaden to approximately twice their normal width. Using mid-positive neuroblasts as a landmark (these remain unchanged in slp mutant embryos), it was possible to characterize the increase in mid expression as an anterior expansion. This aberrant mid expression pattern is unstable; from stage 11 onward mid decays in odd-numbered segments. Conversely, misexpression of slp in the ventral ectoderm from early stage 9 onward led to a complete loss of ectodermal mid expression. These data show that Slp functions as a repressor of mid expression. Taken together with the observation that misexpression of mid in otherwise wild-type embryos results in the loss of Wg expression, these results lead to the conclusion that the Slp-mediated repression of mid anterior to the En/Hh stripe is an important component of wg competence (Buescher, 2004).

As a further test of the relationship between slp and mid, the effect was compared of expressing mid and slp, alone or in combination, on Wg expression. Ectopic expression of mid results in a rapid and almost complete loss of Wg expression, whereas ectopic expression of slp results in weak ectopic expression of Wg posterior to the En/Hh stripe. This slp-induced phenotype resembles that of the loss of mid, except that ectopic Wg expression is weaker and appears randomly in even- and odd-numbered segments. The ectopic Wg expression is blocked when mid and slp are expressed together, suggesting that in this context mid acts downstream of slp. The Wg expression anterior to the En/Hh stripe still decays in UAS-mid/UAS-slp embryos, albeit more slowly and variably than in UAS-mid alone. This result may reflect that Wg expression is sensitive to the amounts of available Mid and Slp. It may also indicate that anterior to the En/Hh stripe, Slp function is required for more than just repression of mid and may possibly have independent activating functions. An analysis of slp1,slp2;mid/H15 quadruple mutants would be highly helpful in clarifying the relationship between slp genes and mid/H15. Unfortunately, the generation of such a quadruple mutant by genetic recombination is impossible because the slp deletion that removes these genes (Δ34B) is on a balancer chromosome that precludes recombination (Buescher, 2004).

The pair-rule modulation of the mid/H15 deletion phenotype results from a different requirement in alternate segments for mid/H15-mediated repression of Wg downstream of Hh signaling. However, in mid/H15 mutant larvae, even-numbered denticle belts also show some defects. This prompted analysis of whether mid/H15 plays a role in the expression of other regulators of late segmental patterning. After stage 11, Wg and Hh signaling regulate other target genes, resulting in the subdivision of each segment into smaller territories. In the posterior, adjacent to the En/Hh-expressing cells, a Rhomboid (Rho)-expressing domain is created. Rho processes membrane bound, inactive Spitz (Spi) to an active, secreted form. Competing Spi and Wg signaling control the decision between naked cuticle and denticle formation, with Spi activating denticle-type specification and Wg specifying naked cuticle. After stage 11, the mid stripe marks the anterior most rows of cells in every segment and colocalizes with rho. In situ hybridization of stage 13 mid/H15 mutants with a rho-specific probe reveals a reduction/absence of rho expression in odd-numbered abdominal segments, suggesting that the excess of naked cuticle in mid/H15 mutant larvae arises from the gain of Wg and the concomitant loss of Spi signaling. The expression of Serrate (Ser), which is normally restricted to cells posterior of Rho in abdominal segments because of repression by Hh signaling, was examined. The Ser domain expands into the Rho domain at the anterior end of the segment in embryos lacking mid, whereas Ser expression is lost completely in embryos with ectopic mid expression. This suggests that Mid acts downstream of Hh signaling to repress Ser in addition to Wg. The anterior expansion of Ser in every segment may contribute to the defects found in even-numbered segments, in which only variable weak ectopic Wg expression is detected (Buescher, 2004).

The data indicate that mid/H15 are negative regulators of Wg and Ser. In the ventral ectoderm of odd-numbered abdominal segments, mid/H15 act to break the symmetry of the Hh-dependent activation of Wg expression. It has been proposed that pair-rule gene activity leaves 'imprints' on all cells and that these imprints predispose cells to express either Wg or En. Slp was found to be such an 'imprint' anterior to the En/Hh stripe, where it predisposes cells to express Wg. The early, pair-rule gene-driven mid/H15 expression appears to be another such 'imprint' that predisposes cells posterior to the En/Hh stripe not to express Wg (Buescher, 2004).

These findings raise several questions. (1) Because the outcome of bidirectional Hh signaling is asymmetric in all abdominal segments, additional factors that prevent the inappropriate expression of Wg in even-numbered segments must exist. At present, such factors are not known. (2) Is the decreased Rho expression in alternating segments a direct consequence of a loss of activation by mid/H15 or a result of negatively acting, ectopic Wg expression? The latter explanation is favored because the ectopic pair-rule-biased expression of Wg corelates with the pair-rule-biased loss of Rho, and ectopic activation of the Wg pathway is sufficient to repress Rho expression posterior to the En/Hh stripe. (3) What is the molecular mechanism of Wg repression by mid/H15? Mid and H15 are members of the T box family of transcription factors and therefore presumably modulate target gene expression directly. The target genes of Mid/H15 are currently unknown. Although it is conceivable that wg is a direct target gene, other scenarios are possible: Mid/H15 may positively or negatively regulate the expression of unidentified genes and thereby modulate Wg or Hh pathway activities, and hyperactivity of either pathway could produce an ectopic stripe of Wg expression. It is noteworthy that a different group of T box genes, the dorsocross genes, has been identified as a negative regulator of Wg expression in the dorsolateral epidermis. However, the Dorsocross target genes are also unknown. Further studies are required to elucidate the mechanisms by which T box proteins negatively regulate Wg expression (Buescher, 2004).

Ancestry-independent fate specification and plasticity in the developmental timing of a typical Drosophila neuronal lineage

In the Drosophila CNS, combinatorial, interdependent, sequential genetic programs in neuroectodermal (NE) cells, prior to the formation of neuroblasts (NBs), determine the initial identity of NBs. Temporal factors are then sequentially expressed to change the temporal identity. It is unclear at what levels this positional and temporal information integrates to determine progeny cell identity. One idea is that this is a top-down linear process: the identity of a NB determines the identity of its daughter, the ganglion mother cell (GMC), the asymmetric division of the GMC and the fate specification of daughter cells of the GMC. Results with midline (mid), which encodes a T-box protein, in a typical lineage, NB4-2->GMC-1->RP2/sib, suggest that at least part of the process operates in GMCs. That is, a GMC or a neuronal identity need not be determined at the NB or NE level. This is demonstrated by showing that Mid is expressed in a row 5 GMC (M-GMC), but not in its parent NB or NE cell. In mid mutants, M-GMC changes into GMC-1 and generates an RP2 and a sib without affecting the expression of key genes at the NE/NB levels. Expression of Mid in the M-GMC in mid mutants rescues the fate change, indicating that Mid specifies neurons at the GMC level. Moreover, a significant plasticity is found in the temporal window in which a neuronal lineage can develop. Although the extra GMC-1 in mid mutants is born ~2 hours later than the bona fide GMC-1, it follows the same developmental pattern as the bona fide GMC-1. Thus, a GMC identity can be independent of parental identity and GMC formation and elaboration need not be strictly time-bound (Gaziova, 2009).

That two cells converge to the same fate from different lineages has been well documented in C. elegans: except in the gut and germline, identical cells in all other tissues originate from multiple lineages. Body wall muscle cells that are almost identical morphologically and physiologically come from four different founder cells. Similarly, identical neurons can be specified by different lineages. For instance, for bilateral neurons among the six sensory neurons involved in mechanosensation, although derived from the same founder cell, their lineages diverge four cell divisions prior to the terminal division. However, Drosophila is not driven by lineages, except for NBs, but they produce distinct progeny lineages specific to a given NB. Therefore, the above conclusion as drawn from studies in C. elegans was not an obvious, or expected, one in Drosophila and this makes the current findings with mid significant. The fate of a cell is not specified simply by a single transcription factor, but instead by a complex combination of cell-autonomous and cell-non-autonomous genetic circuitry. The results indicate that Mid plays a central role in this process in M-GMC, preventing it from becoming GMC-1 of the RP2/sib lineage. Absence of Mid activity initiates a cascade of events in M-GMC that ultimately transforms M-GMC into GMC-1 (Gaziova, 2009).

A NB undergoes multiple self-renewing asymmetric divisions, each time producing a GMC of specific identity, which then generates two neurons of distinct identities. The identity of the first GMC from a NB is dependent upon the gene expression program in the NE cells from which the parent NB is delaminated, and this identity is thought to be invariant. Following division to generate a GMC, the gene expression program in the NB changes so that it produces a second GMC of different and distinct identity from the first GMC. Based on these and several other similar studies, it is currently believed that the identity of a GMC and its neuronal pairs is already determined in the NE and NB levels, i.e. it is ancestry-dependent. However, the current results with mid show that this ancestry-dependent fate specification is not as stringent as once thought, and that the identity of a GMC can be altered without altering the gene expression program in the NB or NE level. Thus, a specific set of neurons (in this case RP2/sib) can be derived or specified from a GMC other than the bona fide GMC by altering the activity of a single gene, in this case mid. One should keep in mind that the ultimate specification of the identity of a GMC (in this case M-GMC/eGMC-1) certainly depends on a complex interplay of many gene products. This study, however, identifies Mid as a key player in preventing M-GMC from becoming GMC-1 of the RP2 lineage. One should also point out that some GMCs, although being generated by different NBs, may have similar potentials and that there might be only one gene responsible for their differences; it is believed that mid as one such gene (Gaziova, 2009).

The results also show that duplication of the RP2/sib lineage, an extensively studied neuronal lineage, can occur by a mechanism or route that is different from those previously described. There are several ways the RP2 lineage can be duplicated. The most common way is through a second NB changing its identity into NB4-2, the parent of the RP2/sib lineage. RP2 lineage duplication can also occur when a GMC-1 divides symmetrically to produce two GMC-1s, each producing an RP2. A GMC-1 can also divide asymmetrically to self-renew and generate an RP2, and the self-renewed GMC-1 divides again to generate another (or more) RP2 or sib. A GMC-1 can also divide symmetrically to generate two RP2s. All these scenarios are different from the one described in mid mutants, in which an unrelated GMC (M-GMC) in a relatively distant location changes its identity to GMC-1 and generates a second set of RP2/sib cells at this distant site. This occurs without changing the expression of any of the genes known to be crucial for fate determination in the precursor NB or NE cells. This has not been observed before and as such adds to the novelty of the results (Gaziova, 2009).

A third set of results that are novel comes from the fact that a second GMC-1-->RP2/sib lineage can be formed 2-2.5 hours after the formation of the bona fide GMC-1-->RP2/sib lineage. This type of plasticity in the timing of formation of a lineage has never been shown before for this or any other lineage in the CNS. There is a certain degree of plasticity in the timing of formation and elaboration of a lineage in the CNS between hemisegments. For example, formation of NB4-2 and its division can be delayed by ~15 minutes between hemisegments. In the case of gsb or en/invected mutants, for example, NB5-3 (which is located close to NB4-2) transforms into NB4-2, thus duplicating the RP2 lineage. NB5-3 (whether transformed into NB4-2 as in these mutants, or not) is formed ~30 minutes prior to the formation of NB4-2. Thus, the sequential production of the duplication can be delayed by as much as 45 minutes in an embryo. A similar interval in the sequential production of the RP2 lineage is also observed in embryos mutant for lottchen (Drop), in which a second NB (possibly NB3-2, located adjacent to NB4-2) changes into NB4-2. The results with mid indicate that an additional GMC-1-->RP2/sib lineage can be formed as much as ~2 hours later than normal for this lineage, and at a site relatively distant from the original location of this lineage. This indicates considerable plasticity in terms of the developmental timing of a neuronal lineage, and that the nerve cord is capable of generating an early forming neuronal lineage also at a later point in time. Moreover, in all previous cases in which a second RP2/sib lineage was formed, it was always formed close to the bona fide RP2/sib lineage. The duplication of the RP2/sib lineage in mid mutants is the first case in which the second lineage is formed at an ectopic site (Gaziova, 2009).

These results are also interesting from another angle. A NB loses it ability, later in development, to produce earlier neurons. In other words, there is a temporally guided progressive restriction on the ability of a NB to generate earlier-born neurons. Indeed, a previous study showed that NBs indeed gradually lose competence to generate earlier-born cells. Although it is not clear whether this is true for all lineages, the current results show that at an organismal level, an earlier lineage can be generated at a later point in development. Thus, whereas the same NB, later in its life, may lose its ability to generate an earlier-born neuron, an earlier-born neuron can still be generated in the CNS at a later point in development, albeit in a different NB or GMC lineage (Gaziova, 2009).

The results show that Mid plays a unique role in preventing M-GMC from becoming GMC-1, ~2 hours after the formation of the bona fide RP2 lineage. It is possible that in the wild type, during evolution a combination of gene expression patterns converged at this ~2-hour time point with the potential to push the M-GMC into GMC-1, but because a nerve cord does not need two RP2s, evolution found a way to prevent this from occurring via expression of Mid in this M-GMC. It is suspected that a similar mechanism might exist in many more lineages than just the M-lineage (Gaziova, 2009).

It has been suggested that the extra cell is not an RP2 neuron. However, this conclusion was based on the observation that this cell does not have an axon projection similar to that of RP2. Since the location of this eRP2 is at the periphery of the nerve cord, one would not expect to observe an ipsilateral projection from this neuron. It was found that the growth cone from this neuron projects anterior and towards the midline, where a choice point for an RP2 projection might exist. This growth cone often fasciculates with the ISN along with the projection from the bona fide RP2. A number of experiments were employed involving different markers, mutant combinations and a very detailed and thorough analysis of this extra lineage. This analysis reveals that it is indeed an RP2: the GMC divides into a larger and a smaller cell akin to the division of the GMC-1 into an RP2 and a sib. One of the two cells, similar to a sib, loses the expression of Eve and does not express RP2-specific markers such as Zfh1. Furthermore, in a mid, insc double-mutant embryo, the esib adopts an RP2 fate, with both cells being the same size and expressing the same RP2 markers as the bona fide RP2 lineage in insc mutants. Similarly, in mid, numb double mutants, both cells become sibs. The two POU genes, pdm1 and pdm2, are required for the specification of GMC-1 of the RP2/sib lineage. In mid, pdm1, pdm2 triple mutants, the eGMC-1 fails to adopt a GMC-1 identity just as the bona fide GMC-1 also fails to adopt a GMC-1 identity (Gaziova, 2009).

However, there are temporal differences in the gene expression pattern between the bona fide RP2/sib lineage and the eRP2/sib lineage. For example, Eve expression begins later in the eRP2 lineage in at least 50% of the hemisegments, as late as subsequent to the eGMC-1 division. Thus, hemisegments are often found with no Eve-positive esib. Since loss-of-function for Eve has no drastic effect on the RP2/sib lineage, this late expression of Eve is likely to be non-consequential to the development of the lineage (Gaziova, 2009).

The bona fide GMC-1-->RP2/sib lineage originates from NB4-2, an S2 NB formed at ~4.5 hours of development (at 22°C). The GMC-1 is formed at 6-6.5 hours of development, although it becomes Eve-positive at ~7 hours of development; it then divides at ~7.45 hours into an RP2 and a sib. The cells undergo a complex migration and then settle within the anterior commissure. An RP2 begins to project its axon growth cone at ~10 hours of development. The eGMC-1 appears to be formed at ~8 hours, becoming Eve-positive at ~9 hours of development. It then divides at ~9.5 hours and begins to project its axon at ~12 hours of development. This indicates that there is significant plasticity in terms of developmental timing as far as the ability of the embryo to generate an RP2 lineage is concerned. All the requisite genetic pathways must still be operational even after 2 hours of development of the bona fide RP2/sib lineage (Gaziova, 2009).

The results indicate that the M-GMC from a row 5 NB (most likely NB5-4) is transformed into GMC-1, as opposed to a NB being transformed into NB4-2. It has previously been shown that in order to specify a NB as NB4-2, that cell should be Gsb-negative. First, 'suppression' results indicate that the eRP2 is generated by a row 5 NB and not a row 4 or 6 NB. However, none of the NBs in row 5 is Gsb-negative in mid embryos; row 5 NBs also had normal expression of three other markers: Wg, Slp and Hkb. This indicates that the identity of these NBs is unlikely to be affected in mid mutants. Second, whereas none of the NBs in row 5 expresses Mid, a row 5 GMC that generates the neuron that transforms into an RP2 in the mutant expresses Mid. It is possible that Mid is expressed in the parent NB of M-GMC but at an undetectable level. However, the conclusion was based on three sets of results: (1) by RNA in situ hybridization using a mid probe, no mid-positive NBs were detected at this location; (2) mid-promoter-lacZ transgenic lines were generatedand the expression of lacZ was basically the same as expression observed with the Mid antibody; and (3) it was possible to rescue/suppress the mid phenotype (i.e. the formation of an extra RP2 lineage) by expressing Mid in M-GMC in mid mutants. Finally, the timing of NB versus GMC specification is also consistent with the conclusion that the transformation occurs at the NB level. It is concluded that a row 5 GMC becomes GMC-1 of the RP2/sib lineage in the absence of wild-type Mid function (Gaziova, 2009).

One issue that was not possible to resolve conclusively is the identity of the parent NB for the M-lineage. The current results indicate that it is NB5-4; the first GMC of this NB gives rise to the M-lineage. Alternatively, it might be NB5-5, in which case the NB has to generate the M-GMC within 1 hour, or it could be a later-born GMC of NB5-3, although based on the position of the M-GMC this latter possibility is unlikely. It was not possible to address the ultimate fate of the M-neuron or its sibling, as to whether they are motoneurons, interneurons or some other cell type (it is unlikely to be glial as they do not express Repo, a glial cell marker), or the function of these cells (Gaziova, 2009).

These results indicate that row 5 NBs are affected in wg mutants, not just rows 4 and 6 as was previously thought. In previous work, a temperature-sensitive (ts) mutant was used and an allele of wg, wgCX4. Whereas the ts mutation is likely to be a hypomorph and retains some Wg activity, wgCX4 is considered a null. However, it was noticed that this allele carries a background mutation(s) that suppresses the wg loss-of-function effect; a partial recombination did eliminate the background suppressor mutation(s) and this 'cleaned up' wgCX4 mutation in trans to another allele of wg, wgIG22, did have the missing row 5 NB defect. It is believed that because of the effect of wg mutation on row 5 NBs, the wg phenotype is mostly epistatic to the mid phenotype in wg, mid double mutants in terms of the extra RP2 lineage defect (Gaziova, 2009).

The T-box-binding element (TBE) was first defined as a 20-bp degenerate palindromic sequence with the highest affinity for the Brachyury protein. However, analysis of the actual target genes reveals that the TBE is highly variable in sequence, number and distribution within their promoters. In the current experiments, with the consensus TBE only the Org-1 protein showed strong activation of the reporter gene, whereas Mid or H15 showed only an ~2-fold increase in transcriptional activation over the GFP control. However, with the gsb-n promoter, which contains a degenerate palindromic TBE sequence, activation by Org-1 was only slightly greater than that by the control protein. By contrast, there was a significant level of activation (~4-fold that of the control) by H15 from the same promoter element (H15 shares 62% identity with Mid), and the level of activation by Mid was ~1.5-fold that of the control, which is slightly more than the stimulation by Org-1. That Org-1 behaves differently to Mid and H15 is consistent with the fact that Mid and H15 belong to the Tbx20 subfamily, whereas Org-1 belongs to the Tbx1 subfamily. This result also shows that although these proteins are all in the Tbx family, they diverge significantly in their sequence preference with regard to the activation of transcription. The Tbx family of proteins is also known to repress transcription. Whereas the Tbx domain binds to DNA, albeit with different specificities according to variations in DNA sequence in the binding site, the rest of the protein is likely to be responsible for either activation or repression (Gaziova, 2009).

Midline governs axon pathfinding by coordinating expression of two major guidance systems

Formation of the neural network requires concerted action of multiple axon guidance systems. How neurons orchestrate expression of multiple guidance genes is poorly understood. This study shows that Drosophila T-box protein Midline controls expression of genes encoding components of two major guidance systems: Frazzled, ROBO, and Slit. In midline mutant, expression of all these molecules are reduced, resulting in severe axon guidance defects, whereas misexpression of Midline induces their expression. Midline is present on the promoter regions of these genes, indicating that Midline controls transcription directly. It is proposed that Midline controls axon pathfinding through coordinating the two guidance systems (Liu, 2009).

To address how Mid activates expression of the three axon guidance genes, the binding sequence of Mid was determined using an in vitro binding site selection method. Mid-binding sequence was selected from a pool of random oligonucleotides using Mid protein affinity-purified from an embryonic extract. The consensus sequence deduced from the selected oligonucleotides was (G/A/T)NA(A/T)N(T/G)(A/G)GGTCAAG. This sequence was found in the upstream regions or an intron of slit, frazzled, and robo, and all of these sites were conserved among several Drosophila species. To determine whether Mid binds to these regions in vivo, chromatin immunoprecipitation (ChIP) was performed using anti-Mid antibody. In all three genes, Mid was present around the Mid-binding sites, but not on regions without the binding site. In contrast, a potential Mid-binding site 32-kb upstream of the commisureless gene, whose expression is not affected in mid mutants, was not occupied by Mid (Liu, 2009).

The importance of the Mid-binding sites in frazzled and slit was assessed by transgenic reporter assays. To test the role of the Mid site in frazzled, reporter genes were constructed that contain the transcription start site of frazzled and an upstream region including a wild-type Mid-binding site (fraPlacZ) or a mutated site (fraMPlacZ). Compared with the wild-type reporter gene, the reporter with a mutated binding site showed reduced expression levels (33% reduction). Thus Mid-binding site is indeed required for the proper expression of frazzled. Mutating the Mid-binding site in slit also caused a severe effect on slit expression. The lacZ expression in sliPlacZ is driven by the slit regulatory element and the endogenous promoter. While sliPlacZ with the wild-type binding site recapitulated the slit expression in the midline glia and lateral cells, base substitutions in the Mid-binding site in sliMPlacZ abolished the lacZ expression. It is possible that the Mid-binding site resides in an essential promoter element of slit, and hence, the base substitutions abolished slit transcription in all cells. However, the same results were obtained using sli4.5HHlacZ and sliM4.5HhlacZ in which the slit regulatory element is fused to a heterologous hsp70 promoter. Since mid was expressed in the lateral cells but not in midline glia, these results suggest that Mid-binding sites in slit control slit transcription via binding to multiple factors: Mid in lateral cells and unknown factor(s) in midline glia. Taken together, these results demonstrate a direct role for Mid in the regulation of frazzled and slit, and suggest that Mid governs the expression of multiple axon guidance genes through directly binding of the Mid sites in their regulatory regions (Liu, 2009).

This study has shown that Mid directly controls transcription of key components of the two major axon guidance systems: the Netrin/Frazzled system and the Slit/ROBO system. Because these two systems are considered to have opposing outputs, it is interesting that the expression of both systems are induced by the same transcription factor, Mid. Dynamic expression of Frazzled and ROBO is required for growth cones to simultaneously respond to both attractants and repellents, integrate these signals, and then respond to the relative balance of forces. These molecules also provide nonautonomous functions required for cell motility, such as mediating cell adhesion and promoting axon elongation. The coordination of axon guidance systems by Mid may thus ensure cooperative actions of multiple guidance molecules in growth cone dynamics, axonal adhesion, and elongation. The role of Mid in the transcriptional regulation of axon guidance might be a conserved function, because its orthologs of human, mouse, and zebrafish Tbx20 are also expressed in motor neurons (Liu, 2009).

JAK/Stat signaling regulates heart precursor diversification in Drosophila

Intercellular signal transduction pathways regulate the NK-2 family of transcription factors in a conserved gene regulatory network that directs cardiogenesis in both flies and mammals. The Drosophila NK-2 protein Tinman (Tin) was recently shown to regulate Stat92E, the JAK/Stat pathway effector, in the developing mesoderm. To understand whether the JAK/Stat pathway also regulates cardiogenesis, a systematic characterization was performed of JAK/Stat signaling during mesoderm development. Drosophila embryos with mutations in the JAK/Stat ligand upd or in Stat92E have non-functional hearts with luminal defects and inappropriate cell aggregations. Using strong Stat92E loss-of-function alleles, this study shows that the JAK/Stat pathway regulates tin expression prior to heart precursor cell diversification. tin expression can be subdivided into four phases and, in Stat92E mutant embryos, the broad phase 2 expression pattern in the dorsal mesoderm does not restrict to the constrained phase 3 pattern. These embryos also have an expanded pericardial cell domain. The E(spl)-C gene HLHm5 is shown to be expressed in a pattern complementary to tin during phase 3, and this expression is JAK/Stat dependent. In addition, E(spl)-C mutant embryos phenocopy the cardiac defects of Stat92E embryos. Mechanistically, JAK/Stat signals activate E(spl)-C genes to restrict Tin expression and the subsequent expression of the T-box transcription factor H15 to direct heart precursor diversification. This study is the first to characterize a role for the JAK/Stat pathway during cardiogenesis and identifies an autoregulatory circuit in which tin limits its own expression domain (Johnson, 2011).

tin expression can be divided into four distinct spatial-temporal phases. Phase 1 tin expression initiates after gastrulation during which Twist (Twi) activates pan-mesodermal tin expression via the enhancer tinB. Phase 2 begins after FGF-mediated mesoderm spreading in which Dpp signals produced by the dorsal ectoderm maintain tin expression throughout the dorsal mesoderm via a second enhancer, tinD. It is during phase 2 that Tin specifies the major dorsal mesoderm derivatives. Phase 3 initiates after dorsal mesoderm progenitor specification and is characterized by a pronounced restriction of tin to the cardiac and visceral muscle progenitors. Upd and Upd2 are expressed in the ventral ectoderm during the transition from phase 2 to phase 3 expression. Phase 4 initiates after precursor specification and is characterized by further restriction of tin to the cardiac precursors that give rise to the contractile cardiomyocytes and the noncontractile pericardial nephrocytes. Phase 4 expression further directs heart cell diversification and maturation and is dependent on a third enhancer element, tinC (Johnson, 2011 and references therein).

To test the hypothesis that the JAK/Stat pathway functions in the cardiac-specific gene regulatory network, a systematic characterization was performed of JAK/Stat signaling during mesoderm development. The JAK/Stat pathway regulates final cardiac morphology as well as heart precursor diversification. Stat92E loss-of-function analysis identified a discrete function for the JAK/Stat pathway in restricting tin during the transition from phase 2 to phase 3 expression. In addition, Stat92E embryos have an expanded pericardial cell domain arguing that the JAK/Stat pathway regulates tin to ensure proper heart precursor diversification. Mechanistically, it was found that the E(spl)-C gene HLHm5 is expressed in a complementary pattern to tin during phase 3 expression and that the JAK/Stat pathway activates HLHm5 expression in the dorsal mesoderm. The E(spl)-C genes in turn repress twi expression to preserve cardiac morphology and restrict tin and H15 expression to direct heart precursor diversification. These findings provide the first evidence of a role for the JAK/Stat pathway in cardiogenesis and identify a novel tin autoinhibitory circuit involving Stat92E and E(spl)-C (Johnson, 2011).

Stat92E is a direct Tin target gene during phase 2 expression; however, Stat92E is expressed in segmented stripes at this stage whereas tin is expressed throughout the dorsal mesoderm. In addition, embryos lacking only the maternal contribution of Stat92E have mesoderm patterning defects. Tin-regulated Stat92E zygotic transcription is therefore insufficient to coordinate mesoderm development. These data suggest that maternally contributed Stat92E is activated in response to segmented Upd and Upd2 activity, binds the Stat92E locus and co-activates Stat92E zygotic transcription with Tin. In addition, ChIP-chip experiments identified Stat92E binding activity and a conserved Stat92E consensus binding sites (SCBS) within the Tin-responsive Stat92E mesoderm enhancer. It is concluded that Stat92E and tin co-regulate a brief, spatially restricted JAK/Stat signaling event that patterns the mesoderm (Johnson, 2011).

Phase 3 tin expression promotes cell-type diversification and differentiation within the dorsal mesoderm and is indirectly activated by Wg via the T-box transcription factors in the Dorsocross complex and the GATA factor Pannier. A key finding of this study is that the JAK/Stat pathway activates the transcriptional repressor HLHm5 in the dorsal mesoderm to establish phase 3 tin expression. Because the HLHm5 cis-regulatory region lacks a conserved SCBS, it is predicted that Stat92E regulates HLHm5 expression through a non-consensus binding site. Alternatively, Stat92E acts at long distances to regulate gene expression. The SCBSs in E(spl)-C could be a platform from which Stat92E regulates multiple E(spl)-C genes that, in turn, regulate HLHm5 expression. In either event, Stat92E-mediated activation of E(spl)-C genes restricts tin in the dorsal mesoderm to establish phase 3 expression. Tin, therefore, establishes an autoinhibitory circuit by activating its own repressors in the JAK/Stat pathway and in E(spl)-C (Johnson, 2011).

Both Stat92E and Df(3R)Esplδmδ-m6 embryos show an increased number of Tin+ pericardial cells and an expanded H15 expression domain. Misexpressing mid or H15 in the mesoderm expands the number of Tin+ cells in the dorsal vessel and embryos misexpressing mid show a phenotype strikingly similar to Stat92E and E(spl) embryos. As mid, and presumably H15, are positively regulated by Tin during St11/12, unrestricted tin expression in Stat92E or Df(3R)Esplδmδ-m6 embryos expands the H15 expression domain. Ectopic H15 then specifies supernumerary Tin+ pericardial cells. Because mid expression is not affected in Stat92E embryos, the expression of mid and H15 must be controlled by distinct mechanisms and might have non-redundant functions (Johnson, 2011).

Although the Twi target genes directing mesoderm development and subdivision have been studied in detail, the molecular mechanism that restricts twi expression after gastrulation remains unclear. One regulator of twi is the Notch signaling pathway, which acts through E(spl)-C genes to restrict twi expression. However, Notch signaling appears to be active throughout the mesoderm after gastrulation. This study suggests that segmented JAK/Stat signaling activity differentially upregulates E(spl)-C gene expression in concert with Notch to produce periodic twi expression in the mesoderm. In addition, pan-mesodermal twi expression causes cardiac defects independently of cell fate specification, suggesting that the cardiac morphology defects in Stat92E embryos are due to dysregulated twi expression. These results highlight a previously unrecognized role for the JAK/Stat pathway and Twi in regulating cardiogenesis (Johnson, 2011).

Pericardial cell hyperplasia without a concomitant loss of cardioblasts has been reported for dpp hypomorphic embryos and lame duck (lmd) embryos. A late Dpp signal, which occurs after the Dpp signal that regulates phase 2 tin expression, instructs the Gli-like transcription factor Lmd to repress Tin expression in fusion competent myoblasts (FCMs). Tin expression appears to expand into the FCM domain in Stat92E embryos; however, the closest Stat92E chromatin binding domain is over 120 kb distal to the lmd transcriptional start site. This study highlights the possibility that sequential JAK/Stat and then Dpp signals regulate Lmd function to direct heart precursor diversification (Johnson, 2011).

In vertebrates, skeletal myogenesis initiates with the periodic specification of somites in the presomitic mesoderm. Cyclical expression of hairy1 in the chick, the hairy- and E(spl)-related family (Her) in zebrafish, and the orthologous Hes family in mice are under the control of Notch-Delta signaling. Loss of her1 and her7 alters the periodicity with which somite boundaries are specified in fish, and artificially stabilizing Hes7 causes somites to fuse in the mouse. Thus, mesoderm segmentation is governed by Notch-Delta regulation of the E(spl)-C genes in both insects and vertebrates indicating that the two processes share molecular homology. A cell culture model of somitogenesis shows that oscillating Hes1 expression is dependent on Stat activity. This study supports the exciting possibility that JAK/Stat signaling and E(spl)-C form a conserved developmental cassette directing mesoderm segmentation throughout Metazoa (Johnson, 2011).



The dorsal vessel forms from bilateral rows of cardioblasts and pericardial cells. The cardioblasts form a line of cells at the leading edge of the dorsal mesoderm. Pericardial cells are arranged in adjacent ventral rows. During dorsal closure, the rows of cardioblasts and associated pericardial cells migrate dorsally and the cardioblasts fuse with their contralateral partners. The two rows of cardioblasts are located medially and fuse to form the lumen of the dorsal vessel and are flanked by rows of noncontractile pericardial cells located ventrally, laterally, and dorsolaterally. H15 (Brook, 1996) and mid (Buescher, 2004) are linked T-box genes that share 89% identity in their T-box domains. The H15-lacZ enhancer-trap (Brook, 1993), inserted 3 kb 5' of H15, shares the expression pattern of both mid and H15 during ectodermal segmentation (Buescher, 2004). Griffin (2000) reported that H15-acZ was expressed in cardioblasts. The mid and H15 expressions in the embryo are first detected in ventral ectodermal stripes and neuroblasts (Buescher, 2004). In the mesoderm, mid is first expressed during early stage 12, where it is detected in two cells per hemisegment in second thoracic through eighth abdominal segments (T2-A8). During stages 12 and 13, the expression pattern evolves into clusters of cells and then into a continuous row of cells in the dorsal mesoderm that fuse dorsally. H15 mRNA is first detected in the dorsal mesoderm in clusters of cells at stage 13, after which H15 is expressed in the same pattern as mid but at lower levels. To confirm that mid and H15 are expressed in cardioblasts, mid and H15 mRNA were examined in embryos carrying an enhancer trap inserted at the seven-up locus (svp-lacZ). svp is expressed in two cardioblasts per hemisegment, and both mid and H15 mRNA colocalize with svp-acZ. As predicted for cardioblast-specific expression, each pair of mid and H15 svp-lacZ-expressing cells is separated by four cells expressing only mid and H15. Thus, the mid and H15 mRNAs are localized to cardioblasts in the mature dorsal vessel (Miskolczi-McCallum, 2005).

Unlike tin and pnr, which are expressed in broad domains throughout the dorsal mesoderm before becoming restricted to a subset of heart cells, mid is initially activated in two cells per hemisegment. To determine where the cells arise, the location of mid-expressing cells was examined in the dorsal mesoderm relative to the ectodermal expression of the segmental markers Wg and Engrailed (En). Wg and En mark adjacent domains in ectoderm overlying the dorsal mesoderm with Wg labeling the row of cells just anterior to the En domain. The two mid-expressing cells arise in similar positions in the underlying mesoderm in each hemisegment. One cell located beneath the Wg stripe and the other lying beneath and slightly to the posterior of the En stripe. As stage 12 progresses, groups of three to six mid-expressing cells are located beneath or near the Wg and En stripes, consistent with the two initial cells dividing and coalescing to form clusters. The clusters fuse to form a continuous line of cells with six cells per hemisegment. The progression of mid expression is consistent with previous mosaic analysis and experiments manipulating the cell cycle in the dorsal mesoderm cells. These studies provided indirect evidence that the six cardioblasts in each hemisegment arise from two progenitors that divide twice to give rise to all six cardioblasts and two of the pericardial cells (Miskolczi-McCallum, 2005).

Previous work has shown that loss of Notch signaling results in a fourfold increase in cardioblasts. To test whether the expression of mid is dependent on the Notch pathway, Notch signaling was blocked with a loss-of-function mutation in the ligand Delta (Dl). Seven to ten mid-expressing cells were observed in each hemisegment in early stage 12 Dlx mutant embryos. Each cluster of mid-expressing cells is associated with an ectodermal En stripe, although clusters are occasionally missing or fused. To ectopically activate the Notch pathway, the intracellular domain of Notch (Nicd) was expressed throughout the mesoderm using the GAL4/UAS system. In twi-GAL4;UAS-Nicd embryos, mid expression in the dorsal mesoderm of stage 12 embryos was often absent. These results suggest that the mid-expressing cardioblast precursors are selected from a single pool of competent progenitors by Notch-mediated lateral inhibition (Miskolczi-McCallum, 2005).

Given the documented pattern of vertebrate Tbx20 expression in heart, expression of Drosophila Tbx20/neuromancer (nmr) was examined in the forming heart. Using fluorescent in situ hybridization, it was determined that both nmr1 (H15) and nmr2 (midline) genes exhibit similar RNA expression patterns late in embryogenesis, but they differ significantly in the onset of expression. Cardiac nmr2 expression initiates at late stage 11 when tinman is restricted to the prospective cardiac mesoderm, whereas nmr1 RNA is only detectable after stage 13. Both persist in an identical pattern as the bilateral myocardial cells begin to assemble into the heart tube at the dorsal midline. nmr2 is first detected at blastoderm stage as well as later in ventral ectoderm derivatives posterior to Wg and overlapping with En/Hh (Qian, 2005).

Since nmr2 is not expressed in the entire tinman-expressing cardiogenic region, fluorescent in situ hybridization of nmr2 double-labeled with various markers of other sets of cardiac progenitors was conducted. Indeed, nmr2 is expressed in a complementary pattern to that of Eve, which marks a subset of pericardial cells. After germ band retraction, the nmr2-expressing cells differentiate into 6 myocardial cells per hemisegment indicated by double labeling with nmrH15-LacZ. The exclusive expression of nmr2 (and nmr1) in all myocardial cells that will give rise to the contractile myocardium suggests that the corresponding cardiac progenitors may co-label with Lbe and Svp, in addition to Tinman. Indeed, double-labeling with Lbe shows that they coincide in two myocardial cells per hemisegment. Cardiac nmr expression (after stage 13) also coincides with other myocardial-specific (e.g., dSUR) but not with pericardial-specific markers (e.g., Zfh-1). However, the possibility cannot be excluded that at earlier stages (11/12) mixed or pericardial-only lineages (e.g., expressing tinman but not eve) transiently express nmr (Qian, 2005).

Defective decapentaplegic signaling results in heart overgrowth and reduced cardiac output in Drosophila: Dpp limits Tin expression, independently of zfh1, by repressing the expression of mid, a stimulator of proliferation

During germ-band extension, Dpp signals from the dorsal ectoderm to maintain Tinman (Tin) expression in the underlying mesoderm. This signal specifies the cardiac field, and homologous genes (BMP2/4 and Nkx2.5) perform this function in mammals. A second Dpp signal from the dorsal ectoderm restricts the number of pericardial cells expressing the transcription factor Zfh1. Via Zfh1, the second Dpp signal restricts the number of Odd-skipped-expressing and the number of Tin-expressing pericardial cells. Dpp also represses Tin expression independently of Zfh1, implicating a feed-forward mechanism in the regulation of Tin pericardial cell number. In the adjacent dorsal muscles, Dpp has the opposite effect. Dpp maintains Krüppel and Even-skipped expression required for muscle development. The data show that Dpp refines the cardiac field by limiting the number of pericardial cells. This maintains the boundary between pericardial and dorsal muscle cells and defines the size of the heart. In the absence of the second Dpp signal, pericardial cells overgrow and this significantly reduces larval cardiac output. This study suggests the existence of a second round of BMP signaling in mammalian heart development and that perhaps defects in this signal play a role in congenital heart defects (Johnson, 2007).

A previous study suggested that a second round of Dpp dorsal ectoderm-to-mesoderm signaling, stimulated by enhancers located in the dpp disk region, initiates during germ-band retraction (stage 12; Johnson, 2003). This is referred to as the second round of signaling because a distinct set of enhancers located in the dpp Haplo-insufficiency (Hin) region activates Dpp dorsal ectoderm-to-mesoderm signaling during germ-band extension (stage 8). Further, the data revealed that dpp dorsal ectoderm expression driven by the Hin region enhancers persists long after germ-band retraction. These studies showed that Hin-region-driven dpp expression is sufficient for Dpp ectodermal functions such as dorsal closure and dorsal branch migration (Johnson, 2007).

Given these data, it appears that the dppd6 inversion prevents the augmentation of dpp expression in the dorsal ectoderm during germ-band retraction that is normally provided by disk region enhancers. The presence of numerous mesodermal phenotypes in dppd6 mutants (Johnson, 2003) suggests that the augmentation of dpp expression is necessary to boost Dpp dorsal ectoderm signals so that they can reach the underlying mesoderm. Perhaps there are barriers of distance or extracellular matrix density between these germ layers that must be overcome (Johnson, 2007).

The data are wholly consistent with the hypothesis that the dppd6 inversion prevents the augmentation of dpp expression provided by disk region enhancers during germ-band retraction. The data further suggest that the augmentation of dpp expression is necessary to boost Dpp dorsal ectoderm signals such that they can reach the underlying mesoderm. Finally, this study shown that during germ-band retraction Dpp signals maintain the boundary between pericardial cells and dorsal muscle cells via two distinct mechanisms: the regulation of gene expression and the restriction of cell proliferation. To regulate gene expression, Dpp signals directly to pericardial cells and restricts Odd and Tin expression in a zfh1-dependent manner. Dpp also limits Tin expression, independently of zfh1, by repressing the expression of mid, a stimulator of proliferation (Johnson, 2007).

With respect to zfh1-dependent regulation, the data support the hypothesis that Dpp restricts Zfh1 expression to regulate the number of pericardial cells derived solely from symmetrically dividing lineages. Lineage analyses have identified both symmetric and asymmetric cell divisions of myogenic and pericardial precursor cells. Pericardial cells are derived from four separate lineages that arise from four distinct precursor cells. Asymmetric precursor cell divisions initiating between stages 8 and 10 give rise to the Odd-positive/Seven up (Svp)-positive pericardial cells and the Eve-positive/Tin-positive pericardial cells (EPCs). In contrast, symmetric division, initiating at the same stage, establishes the Odd-positive/Svp-negative pericardial cells (OPCs) and the Tin-positive/Eve-negative pericardial cells (TPCs). dpp mutations do not affect the number of EPCs or the number of Odd-positive/Svp-positive cells. However, embryos bearing dpp mutations show an increase in the number of OPCs and TPCs. Therefore, the ectopic pericardial cells seen in dpp mutants derive from symmetrically dividing lineages (Johnson, 2007).

Previous reports have shown that regulation of asymmetric cell division is a key mechanism in establishing boundaries among the various cell types in the dorsal mesoderm. For instance, in the absence of Numb, a Notch pathway antagonist, asymmetric progenitor cell division is abrogated and the number of Odd-positive/Svp-positive cells and EPCs increases at the expense of the Svp-expressing cardial cells and Eve-expressing dorsal muscle cells, respectively. This study extends these observations by showing that pericardial cell types derived from symmetrically dividing lineages are also under strict regulatory control (Johnson, 2007).

With respect to zfh1-dependent regulation of pericardial cell number, Dpp restricts cell proliferation and, in turn, Tin expression by limiting mid expression. In wild-type embryos, cell division in the dorsal mesoderm is largely complete by the early stages of germ-band retraction (stage 11), whereas in dppd6 embryos cell proliferation in the dorsal mesoderm continues through stage 13. Interestingly, the number of cells expressing Zfh1 increases from stage 12 to stage 13 in wild-type embryos in the absence of cell division, demonstrating that patterning events subsequent to cell division regulate cell fate choices in the dorsal mesoderm. This hypothesis is supported by the fact that tracing pericardial cell lineages requires inducing mitotic clones by stage 8. Therefore, the ectopically dividing mesoderm cells observed in dppd6 embryos are derived from cells with the potential to become Tin-expressing cells (Johnson, 2007).

During stage 12, tin expression is reactivated in a subset of cardiac cells in a mid-dependent fashion, suggesting that tin expression in precursor cells alone is not sufficient for specifying the ultimate fate of their daughter cells. Moreover, misexpression of mid results in both ectopic cell division and expanded tin expression. Lineage studies support the necessity of reactivating Tin by showing that a single precursor cell gives rise to two Tin-positive/Eve-negative pericardial cells and two siblings that do not express Tin. Thus tin is not reactivated in all subpopulations of pericardial cells. The data suggest that, during stage 12, Dpp prevents tin reactivation in cells occupying lateral regions of the dorsal mesoderm by limiting mid expression (Johnson, 2007).

Development of the dorsal musculature initiates when founder cells are specified in the mesoderm. These founder cells then fuse with neighboring cells to form syncitial myofibers. In the absence of Dpp, the pericardial cell domain expands into the dorsal muscle domain and reduces expression from the dorsal muscle genes Kr and Eve. Since the separation between pericardial and dorsal muscle cells is lost in dpp mutant embryos, it is concluded that Dpp maintains the pericardial-dorsal muscle cell boundary after it is established. Moreover, reducing pericardial cell number increases Kr expression after germ-band retraction, suggesting that cross-repressive interactions between pericardial and dorsal muscle cells contribute to patterning of the dorsal mesoderm. The presence of ectopic pericardial cells in the dorsal mesoderm reduces the number of myofibers comprising the dorsal muscles even though the dorsal muscle founder cells are, for the most part, correctly specified. pMad does not accumulate in Kr-expressing founder cells yet Kr expression is significantly reduced in dpp mutant embryos. Therefore, changes in Kr and Eve expression observed in embryos with altered dpp or zfh1 activity reflect alterations in the number of myoblast fusion events in the dorsal mesoderm (Johnson, 2007).

These data extend a previous study showing that misexpressing Zfh1 reduces dMef2 expression in somatic muscles. This study demonstrates that misexpression of Zfh1 induces ectopic pericardial cells and that the presence of pericardial cells in the dorsal muscle domain reduces myoblast fusion. Therefore, reduced dMef2 expression in embryos misexpressing Zfh1 is likely the result of reduced myoblast fusion and not of direct repression of dMef2 expression by Zfh1. Further, analysis of lmd mutants that have reduced numbers of myoblasts revealed that they also contain an excessive number of pericardial cells. Together, these results suggest that maintaining the pericardial-dorsal muscle cell boundary requires Dpp-mediated cross-repressive interactions between these cell types. Thus, in the absence of Dpp, the transformation of dorsal muscle cells into pericardial cells reduces the number of myoblasts available for fusion (Johnson, 2007).

Experiments in the larvae of Drosophila and other insects suggested that pericardial cells act as nephrocytes that filter the hemolymph. These studies also showed that pericardial cells secrete proteins into the hemolymph, suggesting that one pericardial cell function may be to provide short- or long-range signals. Consistent with this, reducing pericardial cell number reduces heart rate and increases the cardiac failure rate, suggesting that pericardial cells influence the development of cardiac cells (Johnson, 2007).

This study shows that pericardial cell hyperplasia reduces the luminal distance of the heart during systole as well as diastole, resulting in an overall decrease in average pulse distance of each contraction. However, pericardial overgrowth does not alter heart rate, indicating that cardiac cells develop appropriately in the presence of ectopic pericardial cells. Luminal measurements suggest a role for pericardial cells in the mechanics of heart function. One hypothesis for this is based on the fact that pericardial cell hyperplasia results in excess levels of extracellular matrix protein Pericardin (Prc) in the extracellular matrix (ECM) surrounding the heart. Prc is a collagen IV-like ECM protein secreted at high levels from pericardial cells. In dpp mutants, excess Prc is seen predominantly in the posterior of the heart where the pulse-distance reduction was observed. It is proposed that Prc secreted by pericardial cells limits the width of the dorsal vessel at diastole and thus modulates the pulse distance of each heart contraction. Pericardial cell overgrowth would increase Prc deposition, thereby reducing the size of the diastolic heart and the pulse distance. Consistent with this hypothesis, excessive expression of ECM proteins, including collagen IV, was correlated with heart failure in patients presenting with end-stage cardiomyopathy (Johnson, 2007).

It is well documented that many of the early events driving Drosophila embryonic heart development have been conserved in vertebrates. The data provide the first basis upon which to determine if Dpp regulation of Zfh1 or Tin late in heart development is also conserved (Johnson, 2007).

Two orthologs of zfh1, Sip1 and Kheper, have been identified in vertebrates. Zebrafish embryos injected with the Dpp homolog BMP4 show reduced Kheper expression while Xenopus embryos injected with the BMP antagonist Chordin display elevated Sip1 expression. These results suggest the possibility that Dpp repression of zfh1 expression may be conserved in vertebrates. In addition, mammalian Sip1 plays an essential role in heart development. In mice, Sip1 is expressed in neural crest cells (NCCs), paraxial mesoderm, and neuroectoderm. The subset of NCCs that express Sip1 give rise to the septum and large arteries of the heart. Sip1 knockout mice fail to form these NCCs and these mice die midway through gestation with numerous heart defects. Mice lacking the BMP receptors BMPRIA or ALK2 specifically in NCCs also display numerous cardiac phenotypes. In conditional knockout of ALK2 in NCCs, abnormalities are seen in the heart's outflow tract, and conditional knockout of BMPRIA in NCCs results in heart failure and early embryonic lethality similar to Sip1 knockout mice. Thus BMP signals are required for proper specification of NCCs, and loss of BMP signaling in NCCs phenocopies Sip1 knockout mice to an extent. It is tempting to speculate that, as in Drosophila, BMP signals regulate the Zfh1 ortholog Sip1 to correctly specify NCCs and, in turn, to properly pattern the mammalian heart (Johnson, 2007).

With regard to the conservation of late-stage Dpp regulation of Tin, a recent article describing a study of mice with a conditional knockout of Nkx2.5 where expression is missing only during late stages of heart development (post E14.5) is highly relevant. Utilizing rescue of Nkx2.5 mutant embryos with BMP-signaling-pathway components, the study identified a direct connection among BMP4 signaling, Nkx2.5 activity, and heart cell proliferation. Since Nkx2.5 is the Tin homolog, BMP4 is the Dpp homolog, and the mutant phenotype (heart cell hyperplasia) is the same in both species, this suggests that this aspect of Dpp signaling is conserved in mammals. Together with this study, these results suggest that defects in late-stage BMP signaling may play a role in congenital heart defects (Johnson, 2007).

Determination of EGFR signaling output by opposing gradients of BMP and JAK/STAT activity

A relatively small number of signaling pathways drive a wide range of developmental decisions, but how this versatility in signaling outcome is generated is not clear. In the Drosophila follicular epithelium, localized epidermal growth factor receptor (EGFR) activation induces distinct cell fates depending on its location. Posterior follicle cells respond to EGFR activity by expressing the T-box transcription factors Midline and H15, while anterior cells respond by expressing the homeodomain transcription factor Mirror. This study shows that the choice between these alternative outputs of EGFR signaling is regulated by antiparallel gradients of JAK/STAT and BMP pathway activity and that mutual repression between Midline/H15 and Mirror generates a bistable switch that toggles between alternative EGFR signaling outcomes. JAK/STAT and BMP pathway input is integrated through their joint and opposing regulation of both sides of this switch. By converting this positional information into a binary decision between EGFR signaling outcomes, this regulatory network ultimately allows the same ligand-receptor pair to establish both the anterior-posterior (AP) and dorsal-ventral (DV) axes of the issue (Fregoso Lomas, 2016).

This study shows that the choice between two alternative Grk/EGFR signaling outcomes in the follicular epithelium depends on positional input provided by Upd and Dpp. At the posterior, the presence of Upd allows Grk to induce Mid/H15 while, at the anterior, Grk together with Dpp positively regulates Mirr. In this context, Upd and Dpp serve to define the response to Grk/EGFR signaling, since they are not sufficient to induce Mid/H15 and Mirr, respectively, in the absence of Grk (Fregoso Lomas, 2016).

Mutual repression is demonstrated between Mid/H15 and Mirr that is proposed to generate a double-negative feedback circuit that toggles the system between anterior and posterior outcomes. Moreover, in addition to their mutual regulation, analysis of double-mutant clones reveals that Upd and Dpp each regulate both Mid/H15 and Mirr and, thus, each provides input to both sides of this circuit. Upd is required for the expression of Mid and H15 even in the absence of a functional mirr gene, demonstrating that Upd is required for Mid/H15 expression independent of its ability to repress Mirr. Similarly, Dpp signaling can repress Mid independently of its positive effect on Mirr. The choice of Grk/EGFR signaling outcome in this context thus depends not only on mutual repression between these alternate targets but also on their opposing regulation by Upd and Dpp (Fregoso Lomas, 2016).

It is proposed that these elements define a bistable network that controls the choice between two alternative outcomes of Grk/EGFR signaling. These outcomes are irreversible -- e.g., posterior EGFR signaling in later stages cannot induce Mirr unless Mid and H15 are absent - and mutually exclusive, and the factors described in this study include two key elements found in bistable networks: feedback and non-linearity. The feedback in this case is provided by the reciprocal repression between Mirr and Mid/H15, generating a double-negative feedback loop that reinforces the choice of signaling outcome. In addition, bistability requires non-linearity in the response of the circuit to its upstream regulators, which makes the switch more sensitive to graded inputs. It is proposed that, in the follicular epithelium, this is achieved by the joint opposing regulation of the feedback circuit by both Dpp and Upd; each activates one side of the switch while repressing the other, biasing the outcome in the same direction (Fregoso Lomas, 2016).

These alternative responses to Grk are separated in time, as the source of Grk moves from posterior to anterior during the course of development. An important element that determines the choice between them is the early pattern of Mirr expression. In early stages of oogenesis, Mirr is Grk independent and is restricted to the main body follicle cells, due to its repression in the terminal regions by Upd. These main-body follicle cells correspond to the future anterior region of the columnar epithelium, and it is proposed that this early expression of Mirr predisposes them to express Mirr instead of Mid/H15 when Grk adopts its final dorsal anterior localization. Such a role for the early phase of Mirr expression is also consistent with the DV asymmetry of the Mid expression domain; as Grk moves anteriorly, leaving the range of posterior Upd and entering this domain of early Mirr expression and Dpp pathway activity, only the peak dorsal levels of Grk are capable of inducing Mid (Fregoso Lomas, 2016).

These observations also provide an example of how antiparallel signaling gradients can be integrated during epithelial patterning. Tissue patterning by opposing morphogen gradients is observed in developmental contexts as diverse as the Drosophila blastoderm and vertebrate neural tube, where they engage downstream transcriptional networks whose dynamic properties generate reproducible gene expression boundaries. Mutual repression between downstream transcription factors helps define the position and boundaries of cell fate domains, but how the opposing gradients are integrated is not well understood. This study shows that, in the follicular epithelium, the opposing Upd and Dpp gradients are integrated at the level of the Mirr-Mid/H15 feedback circuit. This integration occurs not only at the level of the mutual repression between Mid/H15 and Mirr but also by the ability of each gradient to regulate both sides of this circuit (Fregoso Lomas, 2016).

Together, these elements define the framework of a regulatory network that integrates localized positional information to regulate a binary choice of EGFR signaling outcome. The results allow construction of a model that both accounts for how an individual cell responds to Grk/EGFR signaling and explains how these spatial inputs are integrated across the epithelium to generate a defined pattern of Mid/H15 and Mirr expression, ultimately defining the pattern of the eggshell. Mirr is required for the generation of the high- and low-Broad domains, and Mid/H15 expression is required to define the posterior limit of these domains. The ability of Dpp and Upd to influence the outcome of EGFR signaling allows a single signaling input, namely localized secretion of Grk by the oocyte, to generate multiple distinct outputs that are localized in space and time, thus establishing both the AP and DV polarity of the epithelium and generating a complex and reproducible pattern of cell fates (Fregoso Lomas, 2016).


Because mid and H15 expression coincides with cardioblast fate, tests were performed to see whether mid and H15 are required for cardioblast formation and dorsal vessel development. Because the two genes are very closely linked, a pair of overlapping deficiencies was used to generate embryos deleted for both mid and H15. Df(2L)GpdhA deletes genes beginning with H15 moving proximally, and Df(2L)x528 deletes genes beginning with mid and moving distally (Buescher, 2004). Df(2L)x528/Df(2L)GpdhA (H15 mid null) animals are embryonic lethal and have ventral segmentation defects due to the early essential role that has previously been demonstrated. It is important to note that the expression and effects of mid and H15 in segmentation are restricted to the ventral ectoderm (Buescher, 2004) and thus the loss of mid and H15 does not affect the segment polarity gene expression in the dorsal ectoderm that is required for the proper induction of heart precursors (Buescher, 2004). The region deleted by both deficiencies contains only H15, midline, and a predicted gene of unknown function, CG31467. Embryos were probed with CG31467 anti-sense RNA probes and no hybridization was detected and thus CG31467 is unlikely to contribute to dorsal vessel development (Miskolczi-McCallum, 2005).

To characterize the effects of mid and H15 loss of function on heart development, mutant and control embryos were labeled with antibodies to Tinman. At the end of morphogenesis, each cardioblast pairs with a cardioblast in the opposite row to form the central tube of the dorsal vessel. Four out of six cardioblasts pairs in each segment are labeled with Tin and these can be seen as ordered groups of four pairs of labeled cells separated by two pairs of unlabelled cells. Rows of pericardial cells, marked with Zfh-1 or Zfh-1 and Tin, flank the central rows of cardioblasts. In virtually all H15 mid null embryos, the pairing of the Tinman+ cardioblasts is abnormal with some of the cells pairing and others not in proper alignment. In some cases, the central tube of the dorsal vessel is twisted with one row of cardioblasts lying beneath the other. Pericardial cells are often displaced either laterally or medially from their normal location, possibly as a secondary consequence of defects in cardioblast alignment. The overall disorganization is most severe in the middle of the dorsal vessel, in abdominal segments 3, 4, and 5 (Miskolczi-McCallum, 2005).

In order to test the individual contribution of mid and H15 to the dorsal vessel phenotype, embryos mutant for one gene but not the other were examined. H15x4/Df(2L)GpdhA embryos are deleted for both copies of H15 and one copy of mid (H15 null) and showed no heart defects. In contrast, embryos homozygous for the null mutation mid1 (Buescher, 2004; Nüsslein-Volhard, 1984) have a variable heart phenotype. Typically, 50%-60% of mid1 homozygous embryos exhibit an overall disorganization of the dorsal vessel as indicated by Zfh-1 and Tin double labeling while the rest have fairly normal heart morphology. The phenotypes are similar to but weaker than the phenotype seen in H15 mid null embryos. These results suggest that mid and H15 play critical, partially redundant roles in the morphogenesis of the dorsal vessel with mid having a greater role but with loss of H15 increasing the penetrance and expressivity of the defects. A similar relative requirement has been demonstrated (Buescher, 2004) for mid and H15 in ectoderm segmentation (Miskolczi-McCallum, 2005).

To test whether mid and H15 were required for cell fate specification in the dorsal vessel, the expression of several cardioblast and pericardial cell markers was examined in both wild-type embryos and in H15 mid null embryos. DMef2 encodes a MADS box transcription factor expressed in most muscle cells and is specific to cardioblasts in the heart. In wild-type embryos, Dmef2 marks 52 cardioblasts in each half of the heart. In H15 mid null embryos, the wild-type number of Dmef2 cells is found (51.8, n = 11), indicating that cardioblasts are properly formed in the absence of mid and H15 function. The expression of B2-3-20, a lacZ enhancer trap that marks cardioblasts, and Myosin Heavy Chain (MHC), a marker of muscle differentiation, were also found to be normal in H15 mid null embryos, as was the expression of the Hand mRNA, a marker of all cardioblasts and pericardial cells (Kolsch, 2002). These results suggest that mid and H15 are not essential for cardioblast specification. Pericardial cells were also examined in wild-type and H15 mid null embryos. Zfh-1 labeling was counted in confocal slices from mutant and wild-type embryos to estimate pericardial cell numbers. In segments A2-A8, which are posterior to the lymph gland, similar numbers of Zfh-1-positive cells were found in wild-type (118.5, n = 7) and H15 mid null embryos (121.5, n = 9). The staining of Eve, a marker of two pericardial cells per hemisegment, was also normal in H15 mid null embryos, suggesting that mid and H15 are also not required for the generation of correct numbers of pericardial cells. One difference was noted in the lymph gland, a structure composed of Zfh-1-labeled cells that surround the two anterior-most segments of the dorsal vessel, T3 and A1. An increase in Zfh-1-positive cells was found, with 63.7 cells labeled per gland in H15 mid null embryos compared to 50.9 cells labeled per gland in wild-type embryos. Because mid and H15 are not expressed in the lymph gland primordia, this may be a nonautonomous effect of mid and H15 loss of function in the dorsal vessel (Miskolczi-McCallum, 2005).

The loss-of-function data indicate that mid and H15 are required for heart morphogenesis but are not essential for the specification of cardiac fate. In order to assess the effect of ectopic mid expression on cardiac development, mid was expressed with a UAS-mid construct (Buescher, 2004). Expressing UAS-mid under the control of twist-GAL4 drives mid throughout the mesoderm beginning in gastrulation. An increase in Tin-positive cells was detected in the dorsal mesoderm of twi-GAL4;UAS-mid embryos. By stage 13, a clear increase in the number of Tin-positive cells is visible, suggesting an increase in cardiac fate promoted by the ubiquitous expression of mid in the mesoderm. Similar effects were seen using pnr-GAL4, which drives GAL4 in dorsal mesoderm and dorsal ectoderm, indicating that ectopic mid expression in dorsal mesoderm is sufficient to cause the observed effects (Miskolczi-McCallum, 2005).

The dramatic increase of Tin-positive cells caused by ectopic mid expression in the dorsal mesoderm led to an examination of the effects of ectopic mid expression on visceral mesoderm development, which is also derived from dorsal mesoderm. The expression of Fasciclin III (Fas III), a marker of visceral mesoderm was examined in twist-GAL4;UAS-mid embryos. In many embryos, reduced or gapped expression of Fas III was found, consistent with a loss of visceral mesoderm as a result of ectopic mid expression (Miskolczi-McCallum, 2005).

In addition to the changes in tin and Fas III expression, ectopic mid expression driven by pnr-GAL4 or twi-GAL4 disrupts heart morphogenesis, often preventing the two halves of the heart from fusing and making it difficult to assess how ectopic mid affects specific cardiac lineages. However, when expressing UAS-mid under the control of How-GAL4, another driver with expression throughout mesoderm, similar but weaker effects on Tin and Fas III distribution was observed in stage 13 embryos compared to those observed for twist-GAL4;UAS-mid embryos. Furthermore, a range of defects is seen in older embryos. In some embryos, there is a disruption of heart development similar to that seen with twi-GAL4 and pnr-GAL4, but in most embryos, the two halves of the heart join together but have lateral branches and ectopic cells. The lateral branches contain Tin-positive cells, Zfh-1-positive cells, and Tin-positive Zfh-1-positive cells. The ectopic Zfh-1-positive cells and Tin-positive Zfh-1-positive cells likely represent ectopic pericardial cells. The ectopic Tin-positive cells are likely to be ectopic cardioblasts. To confirm this, β-galactosidase-labeled cardioblasts were examined in How-GAL4;UAS-mid embryos carrying the H15-lacZ enhancer-trap. Ectopic cells labeled with β-galactosidase were observed in branched patterns. In normal H15-lacZ embryos, 52 β-Gal-positive cardioblasts per side were found in the dorsal vessel. In H15-lacZ;How-Gal4;UAS-mid embryos, up to 30 extra cardioblasts were found per half dorsal vessel. Double labeling with Dmef2 further supports the identity of the ectopic cells as cardioblasts. In summary, the loss-of-function data indicate that mid and H15 are not required for fate specification, while the gain-of-function data indicate that ectopic expression of mid in dorsal mesoderm is sufficient to induce ectopic cardiac fate (Miskolczi-McCallum, 2005).

Neuromancer Tbx20-related genes (H15/midline) promote cell fate specification and morphogenesis of the Drosophila heart

To determine the requirement for nmr in Drosophila heart formation, mutants were generated. The nmr1 mutants, nmr1614 and nmr1210, were made by imprecise excision of the H15 P-element located 331 bp 5′ to the transcript start. In both alleles, the 5′UTR, transcription and translation start and part of T-box domain are deleted. In both alleles, no nmr1 transcripts are detected as determined by RT-PCR and whole-mount in situ hybridization, but nmr2 RNA appears unchanged (Qian, 2005).

In order to reduce or eliminate nmr2 function, a transgenic snapback RNA interference (RNAi) approach was taken in conjunction with the UAS-Gal4 system. The cDNA fragment of nmr2 with least homology to nmr1 was used to generate transgenic flies containing UAS-nmr2RNAi. When da-Gal4 is driving nmr2-RNAi ubiquitously, nmr2 RNA is virtually absent. By combining nmr2RNAi transgenes with nmr1 mutants, a quasi-allelic series of loss-of-function was generated for both nmr genes. To distinguish the mesodermal versus ectodermal requirement of nmr, germlayer-specific nmr double mutants were generated. For example, embryos of the genotype nmr1614; 24B-Gal4/UAS-nmr2RNAi (nmr1,nmr2meso−) lack nmr2 RNA in the heart region (Qian, 2005).

nmr1 mutants are viable and exhibit only mild defects in cardiac morphogenesis, and none of the early heart markers seem to be affected. In twi24B-Gal4/UAS-nmr2RNAi (nmr2meso−) embryos, however, eve expression is moderately expanded. Eliminating both nmr genes in the mesoderm (nmr1,nmr2meso−), a more severe phenotype is observed, in that at stage 12, the Eve clusters are dramatically enlarged. Importantly, this expansion appears to be at the expense of lbe expression, which is significantly reduced. Despite the change in the relative proportion of eve and lbe expression in almost half of the double mutant embryos, the extent of cardiac mesoderm delineated by tinman expression is not appreciably altered at this stage. Visceral mesodermal markers also do not seem to be noticeably affected (Qian, 2005).

Consistent with the increase in Eve cell formation with reduced nmr function is the complementary pattern of nmr2 expression relative to Eve positive cells. This raises the possibility that nmr normally antagonizes eve expression, similar to the repression of eve by lbe. In order to test this, nmr was ectopically expressed to see if this causes a reduction in eve expression. Indeed, overexpression of either nmr throughout the mesoderm greatly reduces stage 12 eve expression while expanding lbe. This repression may not (only) be direct, but may (also) be mediated via activation of lbe, for example, which then directly represses eve. The repressive activity of Nmr is consistent with in vitro mouse cell culture studies, in which Tbx20 acts as a repressor rather than an activator (Plageman, 2004). Interestingly, eve is not only repressed by nmr, but eve itself can repress nmr, again consistent with the cross-repressive relationship between eve and lbe. Therefore, it is proposed that nmr plays a crucial role in the distinction of cardiac cell types during the early events of cardiogenesis (Qian, 2005).

After germ band retraction (stage 13/14), the cardiac progenitor lineages have produced a set of contractile myocardial and pericardial cells that will go on to form the mature heart. Since at that stage nmr expression has become restricted to all prospective myocardial cells, their differentiation was examined in nmr mutants, using myocardial- and pericardial-specific markers. Remarkably, even though cardiac-restricted Tinman is not significantly altered at stage 12, by stage 14 myocardial Tinman is dramatically reduced in nmr1,nmr2meso− embryos. The remaining Tinman-positive nuclei often co-label with Eve and are thus of pericardial identity. In addition, staining with muscle-specific transcription factor Dmef2 (normally present in all myocardial but none of the pericardial nuclei) is less or absent in the heart-forming region in these nmr mutant embryos. It is speculated that the reduction in tinman and Dmef2 expression in the forming myocardium is in part due to the loss of lbe expression, which includes the Lbe myocardial progenitors co-expressing tinman. To account for the loss of non-Lbe positive myocardial cells, it is postulated that the Lbe-negative, Tinman-positive myocardial progenitor lineages also require nmr function. Since at stage 12 most if not all cardiac progenitors express tinman and their pericardial and myocardial progeny are not yet distinguishable by position, tinman expression appears normal. It is only later when the lack of myocardial differentiation becomes apparent (Qian, 2005).

Even though the non-myocardial Eve progenitor clusters are enlarged in nmr mutants, the number of progeny Eve pericardial cells (EPCs) is only moderately increased. Another marker of pericardial cells, Odd-skipped (Odd), which does not overlap with Eve, was examined. Indeed, odd expression is also expanded in nmr1,nmr2meso−. This suggests that without nmr function pericardial cell populations are expanded, whereas myocardial differentiation is compromised. Conversely, mesodermal overexpression of nmr diminishes Eve and Odd pericardial cell fates, but increases the number of Dmef2 myocardial cells (Qian, 2005).

Since pannier is able to induce transient ectopic tinman expression in the mesoderm, it was asked if nmr also has that capacity. Indeed, mesodermal expression of either nmr gene causes abundant ectopic tinman expression until about stage 14, but does not persist later. This is consistent with the idea that one of nmr's role is to cooperate with tinman (and pannier, see below) in determining the dorsal-most mesoderm as cardiogenic (Qian, 2005).

The dramatic nmr-induced ectopic tinman expression may also be due to increased proliferation of cardiac progenitors. In vertebrates, for example, it has been shown that Tbx5 can inhibit cell proliferation in vitro. Moreover, loss-of-Tbx20-function in mice results in hypoplastic hearts because of reduced cardiac proliferation. To determine whether nmr affects proliferation in Drosophila, the mitotic marker phospho-histone H3 (pH3) was used. While wildtype embryos exhibit only few proliferating cells in the dorsal mesoderm at stage 13, pan-mesodermal nmr expression increases the number of pH3-labeled nuclei throughout the mesoderm, including the cardiogenic region. At earlier stages (late 11/12), proliferation is also increased compared to wildtype, suggesting that nmr is capable of promoting mesoderm proliferation, in addition to its clear role in cardiac lineage specification. Therefore, it is possible that the loss of myocardial cells in nmr mutants is not only due to misspecification, but because nmr may also be required for myocardial progenitor proliferation. Although the ventral mesoderm also shows excess nmr-induced proliferation, it is unlikely, however, that the cells in that region expressing ectopic tinman originate from over-proliferating (dorsal) myocardial progenitor, since there is no gradient of ventrally migrating cells observed. In order to conclusively address this point, the nmr overexpression phenotype will have to be examined in cell cycle arrest mutants (Qian, 2005).

During the process of dorsal closure, the bilateral primordia of the heart migrate towards the dorsal midline and acquire a polarity that is the result of a typical mesenchyme-epithelium transition. This study has explored whether the morphogenetic mechanism of coordinate myocardial cell alignment and heart tube assembly is controlled by nmr. A large proportion of nmr2meso− or nmr1,nmr2meso− embryos at stage 16/17 exhibit considerable myocardial misalignment defects. This phenotype includes intercalation, gaps, clustering and mis-orientation of the Dmef2-labeled myocardial cells (Qian, 2005).

To explore the cellular mechanism of how nmr regulates myocardial cell alignment, the cytoskeletal architecture and epithelial polarity features of the forming myocardium were monitored. First, the cardiac expression of epithelial polarity markers, namely discs large (dlg), armadillo (arm), crumbs (crb), dmPar6, dystroglycan (dg), α-spectrin and βH-spectrin were examined. Among them, dlg, arm, dg and α-spectrin are expressed in the heart in a stereotyped localized pattern (Qian, 2005).

dlg encodes a MAGUK protein containing three PDZ domains and is required for apical-basal polarity by localizing to the basal-lateral sides of epithelial cells. In myocardial cells of the forming heart tube, however, Dlg protein seems to localize to the dorsal and lateral sides of these cells. Even though the leading edge of the dorsally migrating bilateral rows of prospective myocardial cells may be thought of as 'apical', since they meet at the dorsal midline, the 'new' basal side of this forming heart epithelium is facing the midline and prospective lumen of the heart tube, as is the case for blood vessels in vertebrates. In any case, Dlg is severely mislocalized in myocardial cells of nmr mutants (Qian, 2005).

α-Spectrin also marks the basal-lateral membrane of epithelial cells, and in Drosophila is detected at the dorsal and lateral sides of the myocardium, similar to Dlg. In nmr mutants, α-Spectrin is also mislocalized in the forming heart; but unlike Dlg, α-Spectrin levels are much reduced and frequently non-detectable. The similar phenotype is observed with Arm (Qian, 2005).

Dystroglycan (Dg), a major component of the Dystrophin-Glycoprotein complex, plays a crucial role in the establishment of epithelial polarity of the follicle cells in the Drosophila ovary. Dg strongly accumulates at both the basal and apical sides of myocardial membranes, but is excluded laterally. As with the other polarity markers, Dg localization is dramatically disrupted in nmr mutant embryos. The mislocalization of these polarity genes indicates a failure of the myocardium in proper polarity acquisition. Thus, in addition to their role in cell fate specification, the nmr encoded Tbx20 genes may also coordinate the mesenchyme-epithelium transition and polarized alignment of the myocardial primordia (Qian, 2005).

Since loss-of-nmr-function results in early cardiac specification defects, it is possible that the observed abnormalities in morphogenesis are secondary effects to the early defects. Thus, it was of interest to determine whether or not the early misspecifications due to loss-of-nmr-function can be separated from the later morphogenesis defects. Instead of expressing nmr2RNAi with the pan-mesodermal driver 24B-Gal4, the heart-specific tinCΔ4-Gal4 was used; this drives expression in differentiating myocardial cells as they migrate towards the dorsal midline was used. In tinCΔ4 > nmr2RNAi embryos, early cardiac specification does not seem to be affected, based on Tinman, Eve and Dmef2 staining, nor is dorsal migration or alignment of myocardial cells at the dorsal midline. In contrast, however, epithelial polarity markers are often mis-localized as determined by Dlg, α-Spectrin and Dg staining. Dlg, for example, is no longer detected on the dorsal-lateral sides, but instead is frequently deposited also ventrally or missing on the dorsal myocardial membranes. These data suggest that nmr is required for myocardial cell polarization independently of its role in cell fate specification (Qian, 2005).

The GATA transcription factor Pannier has been shown to be required, along with Tinman, for specification of the heart primordium. pannier null mutant embryos exhibit a reduction of both myocardial and pericardial cell populations, but eve expression is less affected than that of lbe. Thus, the functional relationship between pannier and nmr in cardiac cell type specification was examined. Double-labeling for pannier RNA and nmrH15-LacZ shows that reporter gene expression overlaps with that of pannier at the dorsal edge of the mesoderm. In pannier null mutants, mesodermal nmr expression is completely missing, which is in contrast to the presence of residual tinman and other cardiac marker genes. In addition, pan-mesodermal expression of pannier is sufficient to initiate nmr expression ectopically. In contrast, misexpression of nmr throughout the mesoderm is unable to induce pnr ectopically, and in nmr1,nmr2meso− embryos no detectable change in pannier expression is observed. These findings are consistent with the idea that nmr acts downstream of pannier (Qian, 2005).

To further examine the relationship between nmr and pannier, whether they interact genetically was examined. In pannier mutants, the Eve clusters are moderately reduced, whereas in nmr mutants an increase in the size of these clusters is observed. In contrast, a further Eve cluster reduction is observed in nmr1;pnr double mutants. A similar synergistic enhancement of the pannier phenotype by nmr1 is observed using Tinman as a marker. This is consistent with in vitro data that show a direct interaction between Tbx and Gata factors. However, this also raises the question why nmr1 interacts synergistically with nmr2meso− to enlarge the Eve clusters, whereas with pannier a further reduction of Eve is observed. It is thus possible that during cardiac mesoderm specification nmr not only plays an essential role in the distinction between Eve and Lbe progenitors, but also acts (redundantly) in conjunction with pannier to maintain tinman expression, which in turn is required for maintaining eve expression. In agreement with this interpretation is the observation that nmr overexpression can induce tinman ectopically (Qian, 2005).

Whether nmr expression is regulated by additional cardiac transcription factors was examined. As expected, in tinman null mutants no cardiac nmr expression is observed, since the cardiogenic region is not specified. Mesodermal overexpression of tinman, however, is unable to induce ectopic nmr expression, suggesting other factors may cooperate together with tinman to initiate nmr expression. Indeed, when tinman was overexpressed together with pannier throughout the mesoderm, significantly more ectopic nmr expression was observed than with pannier alone, suggesting that tinman and pannier act synergistically to induce nmr and other cardiac marker gene expression. Dmef2 mutants were also examined. In Dmef2 mutants muscle myosin fails to be expressed. nmr was found to be expressed normally, consistent with Dmef2's role in myocardial differentiation rather than in specification of cardiac progenitors (Qian, 2005).

In summary, nmr1,nmr2meso− embryos, one subpopulation of cardiac progenitors, the Eve cluster, is expanded within the cardiogenic region while another set of cardiac progenitors, the Lbe cluster, is reduced. In addition, nmr genetically interacts with pannier by synergistically aggravating pannier's cardiac phenotype. These findings in conjunction with the timing and pattern of expression suggest that nmr participates, along with tinman and pannier, in the initial specification and cell type allocation within the heart-forming mesoderm (Qian, 2005).

The switch in cardiac cell type specification due to loss-of-nmr-function is reminiscent of the phenotype observed in embryos with increased Hedgehog or Ras signaling, in which lbe expression is eliminated while eve is greatly expanded within the cardiogenic mesoderm. Hedgehog signaling seems to regulate the expression level of the EGF receptor ligand protease, encoded by rhomboid (rho), thus activating Ras signal transduction. This raises the possibility that Nmr may act as a link between ectodermal Hedgehog signaling and cardiac patterning. It would be interesting to find out whether Hedgehog signaling limits the expression or activity of mesodermal nmr, which in turn might antagonize Ras signaling to prevent formation of Eve-expressing progenitors. Interestingly, not only is eve expression abolished upon nmr overexpression, but ectopic Eve can also suppress nmr expression, suggesting that there is a reciprocal antagonism between nmr and eve, as between lbe and eve. It will be interesting to find out if nmr expression is expanded in eve mutants, in which mesodermal eve expression is selectively eliminated (Qian, 2005).

The initially expanded size of the Eve progenitor clusters in nmr1,nmr2meso− embryos does not persist to the same extent after the germ band has retracted and the Eve paracardial cells and muscle founder cells have begun to differentiate. It is possible that from the postulated pre-muscular Eve cluster, although initially enlarged in nmr mutants, a normal number of progenitors are selected. Alternatively, there may be fewer cell divisions by the Eve progenitors, consistent with a possible role of Tbx20 in proliferation. In contrast, another type of emerging pericardial cells, marked by odd expression, remains expanded. These progenitors emerge at a later time than the Eve cells and are possibly subject to a different mechanism of selection and differentiation (Qian, 2005).

The processes of vertebrate cardiac morphogenesis are rather complex and not very well understood beyond a descriptive level. The Drosophila heart, in contrast, is remarkably simple but nevertheless resembles that of vertebrates in its initial assembly from bilateral primordial tissue into a highly organized linear vessel-like tube. It is thus of interest to understand the control mechanisms by which a primitive heart tube forms. The defects in myocardial alignment, heart tube assembly and proper polarity acquisition observed in nmr mutants indicate that these T-box genes play a crucial role during these processes of cardiac morphogenesis. Moreover, when nmr2RNAi is specifically targeted to the differentiating myocardial cells, the overall alignment of the bilateral myocardial primordia at the dorsal midline is little affected but the correct epithelial polarity is not established. Together, these observations further suggest that cardiac determination and distinction of cell types are controlled by mechanisms that are separable and perhaps independent from those that govern heart tube assembly and cellular polarity prerequisite for morphogenesis. It will be interesting to find out what transcriptional targets nmr controls at progressively later stages of heart development, and whether vertebrate Tbx20 also influences multiple steps during heart development (Qian, 2005).

This report provides the first evidence that nmr/Tbx20 also has a role in regulating myocardial polarization, in addition to its role in cardiac cell specification. Studies of cell polarity in other tissues, such as salivary gland, oocyte, neuroblast, suggest cell polarization is important for organelle transport and cell-cell communication via adherens junctions or extracellular matrix components. Thus, a failure in proper polarity acquisition is likely to contribute to the observed disorganization of the heart tube. Since only some of the polarity markers tested exhibit a typical epithelial subcellular distribution in the forming myocardium, it is speculated that there may be significant differences by which the heart epithelium is involved in this organ's morphogenesis. Further explorations into the process of cardiac cell polarization are necessary to obtain a mechanistic understanding of cardiac morphogenesis (Qian, 2005).

Genetic analysis demonstrates a regulatory interaction between nmr and other pivotal factors involved in Drosophila heart development, namely pannier and tinman. For example, loss-of-pannier-function abolishes mesodermal nmr expression, while pan-mesodermal overexpression of pannier results in ectopic nmr expression, which is synergistically augmented by co-expression of tinman. This is consistent with the idea that nmr is a transcriptional target of the cardiac determinants, Pannier and Tinman. Interestingly, the emerging cardiac cell types exhibit a differential susceptibility to pannier; pericardial Eve being the least sensitive, which is consistent with the absence of GATA consensus binding sites in the mesodermal eve enhancer (Qian, 2005).

nmr does not only appear to be a target of pannier but also interacts with pannier genetically in the overall formation of cardiac mesoderm: nmr1;pnr double mutants have a more dramatic reduction in heart-associated cells stained with Tinman or Eve than pannier single mutants (nmr1 single mutants do not exhibit a cell specification phenotype). This indicates that nmr also participates in the initial specification of cardiac competence, but this particular role of nmr is redundant in the presence of pannier, since nmr1,nmr2meso− embryos initially have a normal level of cardiac-restricted tinman expression. This idea is supported by the finding that overexpression of nmr is sufficient to drive ectopic tinman expression. Thus, it appears that nmr has two, in part opposing, roles during early cardiac specification: it acts redundantly with pannier to specify tinman-expressing cardiac mesoderm from which all cardiac cell types emerge, but also acts in distinguishing subpopulations of cardiac progenitors (Lbe versus Eve cell fates), perhaps by negatively mediating positional information provided by the ectodermal Hedgehog signal (Qian, 2005).

Tbx20 exhibits prominent expression in the heart of all vertebrate and invertebrate organisms examined, and phylogenetic analysis places Tbx20 within the Tbx1 subfamily that is distinct from Tbx5, suggesting evolutionary conservation. In Zebrafish, Tbx20/hrT is co-expressed with Nkx2-5 and Gata4 in cardiogenic as well as in non-cardiac mesoderm adjacent to the tail bud (Szeto, 2002). Since a genetic interaction is observed in Drosophila between Tbx20/nmr and Nkx2-5/tinman, Gata4/pannier, it may be that a similar interaction takes place in vertebrates. Morpholino knock-down of Tbx20/hrT in zebrafish apparently affects chamber morphology, which is consistent with observations of morphogenesis defects in Drosophila nmr mutants. Whether Tbx20 plays a role in cardiac cell polarization in vertebrates remains to be determined. In chicken, Tbx20 is expressed in a complementary pattern to Tbx5 in the embryonic ventricles, and in ovo electroporation and cell culture studies suggest an antagonistic relationship between them (Plageman, 2004). This is reminiscent of the complementary expression and antagonism observed between nmr and eve in flies, which lack a Tbx5 homologue. Taken together, these studies suggest that Tbx20 genes are likely to play multiple roles during cardiogenesis, ranging from specification, proliferation and morphogenesis to cardiac physiology (Qian, 2005).

Functions of the segment polarity genes midline and H15 in Drosophila melanogaster neurogenesis

The Drosophila ventral nerve cord derives from neural progenitor cells called neuroblasts. Individual neuroblasts have unique gene expression profiles and give rise to distinct clones of neurons and glia. The specification of neuroblast identity provides a cell intrinsic mechanism which ultimately results in the generation of progeny which are different from one another. Segment polarity genes have a dual function in early neurogenesis: within distinct regions of the neuroectoderm, they are required both for neuroblast formation and for the specification of neuroblast identity. Previous studies of segment polarity gene function largely focused on neuroblasts that arise within the posterior part of the segment. This study shows that the segment polarity gene midline is required for neuroblast formation in the anterior-most part of the segment. Moreover, midline contributes to the specification of anterior neuroblast identity by negatively regulating the expression of Wingless and positively regulating the expression of Mirror. In the posterior-most part of the segment, midline and its paralog, H15, have partially redundant functions in the regulation of the NB marker Eagle. Hence, the segment polarity genes midline and H15 play an important role in the development of the ventral nerve cord in the anterior- and posterior-most part of the segment (Buescher, 2006).

Previous studies of segment polarity gene function have focused mainly on the posterior part of the segment. These studies have revealed an intricate regulatory network involving the wg, engrailed (en), invected (inv), gooseberry (gsb) and patched (ptc) genes underlying the formation and specification of NBs in the posterior part of the segment. By contrast, the formation and specification of NBs in the anterior-most part of the segment are less well understood. This is a study of the segment polarity genes mid and H15, which are expressed in the neuroectoderm (NE) of the anterior- and posterior-most part of each segment. Mutations affecting mid were first identified in a screen for segmentation defects and additional alleles have been identified in screens for axon guidance defects in the peripheral nervous system (lost in space) (Kolodziej, 1995) and the ventral nerve cord (Seeger, 1993). The mid gene was cloned and shown to encode a T-box transcription factor (CG6634). The mid gene is flanked by its paralog H15 (CG6604). This study shows that mid and H15 are largely co-expressed in the NE, several identified NBs and a subset of the putative neuronal and glial progeny of these NBs. mid, but not H15, is required in the NE for the formation and specification of NBs in the anterior-most part of the segment. Furthermore, in individual NBs, mid and H15 have partially redundant functions in the regulation of NB marker gene expression. Accordingly, in the mature ventral nerve cord of midline and H15-deficient embryos, a loss of neurons is found and a misspecification of individual neuronal/glial fates (Buescher, 2006).

The mid RNA expression pattern is typical of segment polarity genes. Briefly, from stage 5 onwards, mid is expressed in 14 stripes, with those in presumptive, odd-numbered abdominal segments being wider and more intensely stained than those in even-numbered abdominal segments. Up to stage 9 mid RNA is co-expressed with En, but also extends two to three rows of cells posteriorly to the En domain in the odd-numbered stripes. During stage 9, these odd-numbered stripes narrow, and from late stage 9 until the end of stage 11, mid colocalizes exactly with the En domain in all stripes. During stage 12, mid expression within the En domain decays and is subsequently re-initiated in a narrow stripe posterior to the En domain; this expression is maintained until the end of embryogenesis. Hence, mid RNA is found in the NE from which early and late born (SI-SV) row 6 and row 7 NBs arise and (in odd-numbered abdominal segments) from which early born (SI-SII) row 1/2 NBs derive (the row of NBs posterior to the En domain is referred to as row 1/2, because it produces row 1 and row 2 NBs). The mid gene is flanked by its paralog H15 which has previously been shown to have partially redundant function with mid. Using an H15-specific RNA probe, it was determined that H15 RNA is first observed in segmentally repeated stripes at stage 7: this is considerably later than mid and initially at a much lower level than mid expression although at later stages, H15 expression levels increase and from stage 9 onwards are comparable to mid expression levels. Like early mid stripes (prior to stage 9), early H15 stripes in presumptive odd-numbered abdominal segments are wider and more intensely stained than stripes in even-numbered segments (Buescher, 2006).

To examine mid/H15 expression in NBs, wt embryos were stained with a mid-specific RNA in situ probe and the viable lacZ enhancer trap H15 was stained with anti–β-galactosidase (βgal). mid-specific RNA in situ studies showed that all NBs that delaminate from mid-positive NE transiently express mid but in most NBs expression rapidly decays. However, two SI NBs and one SII NB accumulate and maintain high levels of mid RNA while a second SII NB maintains lower levels of mid expression. These mid-positive NBs appear posteriorly adjacent to the Wg expression domain. H15-lacZ expression in NBs is indistinguishable from that of mid. To identify the mid/H15-positive SI NBs, double label experiments were performed with an antibody against Worniu (Wor), which is expressed in all NBs. At early stage 9, anti-Wor labels nine SI NBs which can be identified based on their positions and it was possible to identify the mid-positive NBs as NB2-5 and NB7-4. To identify the two SII mid/H15-positive NBs, their positions were mapped along the AP and DV axes relative to the En stripe and the NB marker gene eagle (eg). The results indicate that these SII NBs are En-positive and form medially, adjacent to the Eg-positive NB7-3, identifying the mid/H15-positive SII NBs as NB1-2 and NB7-2. From stage 10 onwards, mid/H15 expression was also found at the ventral midline: time of birth, position and the co-expression of En identify this cell/cell cluster as the MNB or its early progeny (Buescher, 2006).

Expression of mid/H15 in the developing nerve cord increases up to stage 14 when each hemisegment contains approximately 50 (thorax) or 40 (abdomen) mid/H15-positive cells. After stage 14, some mid expression decays so that at the end of embryogenesis (stage 16/17) all hemisegments contain approximately 15 cells which express mid. H15 RNA is observed in a similar pattern. By contrast, at stage 16, H15lacZ expression is found in a larger number of cells, probably reflecting the perdurance of LacZ protein (Buescher, 2006).

Most mid/H15-positive cells are neurons as assayed using the neuron-specific anti-Elav antibody. However, co-expression of H15-lacZ and the glial marker Repo was observed in the dorsal and ventral channel glia and two lateral subperineural glia. These cells also express En, suggesting that they are identical to the previously described En-positive glial progeny of NB7-4 (Buescher, 2006).

mid/H15 and En are co-expressed in NBs 1-2, 7-2 and 7-4. However, co-expression in most of their progeny is transient and in the stage 16 lateral nerve cord is restricted to two to three cells, including the lateral subperineural glia. A similar situation was found at the ventral midline: the MNB stains positively for mid RNA/H15lacZ and En protein and at stage 14 the En-positive progeny of the MNB express mid/H15. However, mid/H15 expression subsequently decays and at stage 16 mid/H15 expression at the ventral midline is restricted to the dorsal and ventral channel glia. Notably, mid/H15lacZ expression was never observed in the midline glia which derive from the region anterior to the En domain (Buescher, 2006).

This analysis of the mid/H15 expression patterns demonstrates that these genes are co-expressed in the NE from which row 6, row 7 and early row 1/2 NBs arise. mid/H15 are also co-expressed in the MNB, NB7-2, NB7-4, NB1-2 and NB2-5 and at least a subset of their progeny. Due to the lack of appropriate markers, the lineages of NB7-2, NB1-2 and NB2-5 have remained among the least studied lineages in the VNC. The observation that these NBs are part of a very small group of mid/H15 expressing NBs will greatly facilitate the analysis of their respective lineages (Buescher, 2006).

To investigate the roles of mid/H15 in the development of the VNC, the mutant phenotypes were studied associated with mid and/or H15 loss-of-function alleles. In this study, the alleles midGA174, mid1, mid2 and H15x4, and a combination of the deficiencies GpdhA and x528 were used. midGA174 and mid1 are EMS-induced, putative amorphic alleles, while mid2 is an EMS-induced strong hypomorphic allele. H15x4 is a deletion of the H15 locus and the combination of deficiencies x528 and GpdhA deletes only mid, H15 and one additional predicted gene (CG31647) of unknown function (Buescher, 2006).

During embryonic stages 9-11, when NB formation takes place, mid/H15 RNA is expressed in the NE from which row 6, row 7 and early row 1/2 NBs arise. To determine whether mid/H15 play a role in the formation of these NBs, mid and H15 single mutant and mid/H15-deficient embryos were examined with the anti-Wor antibody which labels all NBs. At early stage 9, Wor expression in wt embryos is found in four NBs which arise from mid/H15-positive NE: NB1-1 and NB2-5 (row 1/2), and NB7-1 and NB7-4 (row 7). Staining of early stage 9 midGA174 mutant embryos revealed a frequent loss of Wor expression in row 1/2 NBs, with a strong bias towards odd-numbered abdominal segments: for example, NB1-1 was lost in 68.7% of the odd-numbered abdominal segments compared to only 5% loss in even-numbered abdominal segments. NB formation was also affected at the NB2-5 position, albeit less severely; for example: in midGA174 mutant embryos, NB2-5 was lost in 32% of the odd-numbered and 7% of the even-numbered abdominal segments. By contrast, formation of early row 7 NBs was found to be unaffected in midGA174 mutant embryos. Staining of stage 9 mutant embryos with an antibody against Ase, an additional marker for all delaminated NBs, confirmed these results. The neuronal marker gene even-skipped (eve) is not expressed in NBs, but in stage 9 wt embryos, Eve protein is present in the first-born progeny of the SI NBs 7-1 and 1-1; thus the presence of Eve-positive GMCs represents a read-out of NB formation within row 7 and row 1/2. Staining of stage 9 midGA174 mutant embryos revealed that GMC7-1a formed normally but GMC1-1a was frequently absent in odd-numbered segments. This supports the observations that (1) mid function is required for SI NB formation in row 1/2 predominantly in odd-numbered segments and (2) that mid is dispensable for SI NB formation in row 7 (Buescher, 2006).

Previous studies have shown that while some segment polarity genes control the formation of early- and late-born NBs within their activity domain (for example gsb), others are only required for late forming NBs. To investigate whether mid plays any role in the formation of later-born NBs (SII-SV), mid mutant embryos were stained with markers which label either all NBs or identify subsets of NBs. In late stage 11 wt embryos, all NBs have delaminated and form a continuous two-dimensional sheet. In midGA174 mutant embryos, a frequent loss of Wor expression, predominantly in odd-numbered abdominal segments, results in a fragmented appearance of the NB sheet. This indicates that early and late-born NBs are lost in mid mutant embryos. To determine if the loss of mid affects the formation of late-born NBs in rows 6 and 7, NBs were labeled with an antibody against En, which is at least transiently present in all row 6 and row 7 NBs and the MNB. Staining of stage 11 midGA174 mutant embryos did not reveal any loss of En-positive NBs in the lateral CNS indicating that the requirement for mid in NB formation is restricted to row 1/2. However, occasionally a loss/reduction of En expression was observed in the position of the MNB, which forms slightly posterior to the En stripe. This loss/reduction occurred almost exclusively in odd-numbered abdominal segments. The conclusion that mid function is not required for NB formation in rows 6 and 7 was corroborated by examination of the NB marker gene eg. In wt embryos, Eg is expressed in NB7-3, NB6-4, NB2-4 (which all derive from mid/H15-positive NE) and in NB3-3. In mid mutant embryos, a loss of Eg expression was occasionally observed in NB2-4 position (17.8% loss) but not in the NB6-4 and NB7-3 positions. As seen for other row 1/2 NBs, loss of NB2-4 occurred predominantly in odd-numbered abdominal segments. Finally, the formation was monitored of the anterior glioblast which arises in the lateral-most position of row1/2. Staining of stage 10 mid mutant embryos with the glial marker anti-Repo revealed that the glioblast forms normally (Buescher, 2006).

The loss-of-function phenotype of mid with respect to NB formation is only partially penetrant in midGA174 embryos. The most severely affected progenitor cell, NB1-1 in odd-numbered abdominal segments, fails to form in 68.7% of the hemisegments. In 31.3% of the odd-numbered hemisegments, an NB still forms and expresses Wor and Ase. Consistent with the notion that midGA174 and mid1 are amorphic alleles, no significant increase in the loss of NB1-1 was found in midGA174/deficiency or mid1/deficiency hemizygotes. This raises the question of whether the mid paralog H15 can compensate for the requirement for mid. Deletion of the H15 gene alone (H15x4) had no effect on the NB formation. Moreover, removal of both copies of the mid and H15 genes did not appreciably enhance the loss of NBs indicating that H15 plays no role in NB formation even in the absence of mid (Buescher, 2006).

In summary, the results show that mid, but not H15, plays an essential role in the formation of early- and late-born row 1/2 NBs predominantly, but not exclusively, in odd-numbered abdominal segments. This requirement for mid reflects the neuroectodermal expression of mid posterior to the En domain and its segmental differences. Earlier studies had indicated that an additional segment polarity gene, patched (ptc), is required for the formation of row 1/2 NBs. This raises the question of whether mutual regulatory interactions between mid and ptc underlie the requirement for both genes. Loss of mid does not result in changes of the ptc expression pattern, and mid expression is not affected by ptc mutants. Mutation of either mid or ptc alone does not result in a complete loss of NB formation in row 1/2, suggesting that these genes may have partially redundant functions in NB formation. However, in mid/ptc double mutants, odd-numbered segments are shorter than in wild-type embryos by approximately two rows of cells in the neuroectoderm posterior to the en domain (the region from which row 1/2 NBs delaminate). Thus, mid and ptc appear to have redundant functions in maintaining the integrity of the neuroectoderm. This early defect makes a proper interpretation of subsequent events in NB formation impossible (Buescher, 2006).

The defects in NB formation in mid mutant embryos do not reflect the neuroectodermal expression of mid within the En domain where mid function is dispensable. This apparent lack of mid function may be explained by a functional redundancy of individual segment polarity genes with respect to NB formation. A functional redundancy of the hh and wg genes in the formation of NB7-3 has been reported and it is conceivable that segment polarity genes with expression domains overlapping that of mid, such as en and hh, may compensate for the loss of mid. Although it has long been established that loss of segment polarity gene function results in the loss of NBs, the molecular mechanisms by which segment polarity genes promote NB formation have remained unclear. Further studies are required to determine regulatory interactions between the segment polarity genes and the proneural genes of the AS-C and the NB-promoting gene SoxNeuro (Buescher, 2006).

Individual NB identity is specified in the NE by the overlapping expression of segment polarity and dorso-ventral patterning genes; loss of segment polarity gene function results in misspecification of NBs along the antero-posterior axis. mid functions in the NE to break the symmetry of Hh-signaling and loss of mid results in ectopic neuroectodermal Wg expression posterior to the En/Hh domain. In wt embryos, Wg-positive NE gives rise to the row 5 NBs, all of which are Wg-positive at the time of birth. To examine whether the ectopic neuroectodermal expression of Wg results in ectopic Wg expression in NBs, stage 11 mid mutant embryos were stained with the anti-Wg antibody. Wg-positive NBs were observed in row 1/2 predominantly in odd-numbered segments. Ectopic Wg in NBs was only partially penetrant and highly variable with respect to the DV position (identity) of the affected NBs, most probably reflecting the substantial loss of NBs in this region. Removal of both copies of mid and H15 did not enhance the severity of the phenotype. These data show that mid, but not H15, is required to prevent the formation of Wg expressing NBs in row 1/2 in odd-numbered abdominal segments (Buescher, 2006).

Row 1/2 NBs are distinguished from NBs in adjacent rows by the expression of Mirror (Mirr). Mirr expression is initiated during stage 7 in transverse stripes in the NE. Across the ventral midline, the neuroectodermal expression of Mirr extends further anteriorly by 2-3 rows of cells. All row 1/2 NBs, the MNB and NB6-1 are at least transiently Mirr-positive. Staining of stage 7 midGA174 mutant embryos revealed a strong reduction of neuroectodermal Mirr expression. Loss of Mirr expression is more pronounced in odd-numbered segments. In stage 10 midGA174 mutant embryos, a strong loss of Mirr is observed in the row 1/2 NB layer predominantly in odd-numbered abdominal segments; this loss arises through the additive effects of the loss of Mirr expression in the NE (which occurs prior to NB formation) and the general loss of row 1/2 NBs. The severity of the phenotype was not enhanced in mid/H15-deficient embryos. It is noteworthy that the loss of Mirr function itself (mirrcre2) does not result in defects in NB formation suggesting that mid does not promote NB formation via Mirr. In summary, Mirr expression represents a further aspect of row 1/2 NB identity and mid, but not H15, contributes to the specification of this identity by promoting the expression of Mirr in odd- and in (to a lesser extent) even-numbered abdominal segments. The consequences of the loss of Mirr function in NB specification and lineage elaboration are as yet unknown (Buescher, 2006).

To investigate whether loss of mid/H15 results in additional misspecifications of NBs, midGA174 mutant embryos were stained with the anti-Eg antibody. In wt, mid/H15 and Eg expressing NBs are close neighbors but expression of these genes appears mutually exclusive. In mid mutant embryos, ectopic Eg expression was found in NB7-4 (12%), in both odd- and even-numbered abdominal segments. By contrast, removal of the H15 gene alone did not result in ectopic Eg expression. However, removal of both copies of mid and H15 caused an enhancement of the phenotype (37.8%) indicating that in the absence of mid, H15 contributes to the regulation of Eg expression. These results indicate that both mid and, to a lesser extent, H15, act as negative regulators of Eg expression (Buescher, 2006).

A role for mid in the negative regulation of Eg expression was surprising since in wt embryos, two of the Eg-positive NBs (NB6-4 and NB7-3) derive from mid/H15-positive NE. However, in contrast to NB7-4, NB6-4 and NB7-3 rapidly lose mid/H15 expression during NB formation while mid/H15 expression in NB7-4 is not only maintained but also upregulated. These results suggest that, in NB7-3 and NB6-4, mid and Eg expression occurs consecutively while prolonged expression of mid in NB7-4 prevents Eg expression. To test this hypothesis, the expression of mid was extended in NB6-4 and NB7-3 using enGal4 to drive the expression of mid. Staining of stage 11 embryos with anti-Eg revealed a complete loss of Eg expression in the NB6-4 and NB7-3 positions. In addition, ectopic expression of mid with the pan-neural driver scabrousGal4 (scaGal4;UAS-mid) resulted in a nearly complete absence of Eg expression. These results confirm that high levels of mid negatively regulate the expression of Eg in NBs. The ectopic expression of Eg in mid mutant embryos is restricted to NB7-4, indicating that the absence of mid per se is not sufficient to permit Eg expression. The regulation of Eg expression involves positive acting factors such as Huckebein and En which have locally restricted expression patterns. The finding that mid negatively regulates Eg in NB7-4 indicates that Eg expression in individual NBs is achieved by a combinatorial code of positively and negatively acting factors (Buescher, 2006).

In segment polarity gene loss-of-function mutants, NBs are often misspecified, such that NBs which form in one particular row adopt the identity of NBs which develop in an adjacent row. Earlier studies have suggested that row 3/4 NB specification versus row 1/2 specification is accomplished by Wg signaling, which is thought to be high in row 3/4 and low or absent in row 1/2. Consistent with such a role for spatially restricted Wg signaling, ectopic Wg expression seems sufficient to induce row 1/2 NBs to acquire row 3/4 identities. In mid mutant embryos, ectopic Wg expression in the NE of row 1/2 appears at stage 9, the time from which SII to SV NBs form. This prompted an examination of whether loss of mid function causes row 1/2 NBs to adopt a row 3/4 identity. To score row 3/4 identity, the neuronal marker protein Eve was examined. Eve is expressed in the progeny of two row 3/4 NBs: the RP2 neuron which derives from NB4-2 and the EL neuron cluster, which derives from NB3-3. Staining of stage 16 midGA174 mutant embryos did not show ectopic EL neuron clusters, indicating that there is no duplication of a NB3-3 identity. However, in mid mutant embryos, a gain of Eve expression was observed in one neuron per hemisegment, located at an antero-posterior position similar to that of RP2. This extra Eve-positive cell is located lateral to the wt RP2 cell and was found in 46% of the hemisegments with no preference for odd-numbered segments. Removal of both copies of mid and H15 resulted in a strong enhancement of the phenotype, indicating that in the absence of mid, H15 plays a role in the restriction of Eve. Using an eveGal4 driver to direct tau-lacZ expression, it was established that the extra Eve-positive cell projects its axon towards the ventral midline, in contrast to the wt RP2 motorneuron, which has a lateral axon projection which exits the VNC. Hence, this cell is not an extra RP2 neuron and does not indicate a duplicated NB4-2 lineage. However, its parental NB could not be identified (Buescher, 2006).

No evidence was found for row 1/2 NBs adopting row 3/4 identities in mid mutant embryos although it is conceivable that ectopic Wg expression in mid mutant embryos occurs too late (stage 9) to cause a change of NB identities (Buescher, 2006).

To assess defects in the mature VNC, mid/H15 mutant embryos were examined with the anti-En antibody. At stage 16 of embryonic development, each wt hemisegment contains three clusters of cells with high levels of En expression: a small cluster of 2-3 En-positive cells at the lateral-most border of the VNC, a larger cluster comprising 5-6 cells is located in a medio-lateral position and 5-7 En-positive neurons and the channel glia at the ventral midline. mid/H15 are co-expressed with En at different stages in neurogenesis and hence loss of mid/H15 may affect NB specification, lineage elaboration and neuronal/glial differentiation. Staining of stage 16 midGA174 mutant embryos with the anti-En antibody revealed a severely aberrant En expression pattern. A frequent loss of the lateral-most En-positive cell clusters was observed, while the En positive medio-lateral cluster contained fewer cells (3-4 instead of 5-6) and the cells were spatially disarranged. At the ventral midline, the number of the En-positive neurons was reduced to 4-5 cells in most segments and completely absent in 12% of the segments. The channel glia frequently showed migration defects or were absent. In H15 single mutant embryos (H15x4), no loss of En-positive neurons was found. However, removal of both copies of mid and H15 enhanced the severity of the mid single mutant phenotype. Defects were observed with similar frequencies in odd- and even-numbered segments reflecting the observation that mid/H15 expression in NBs and their progeny shows no segmental differences (Buescher, 2006).

In summary, mid and, to a lesser extent, H15 play an important role in the formation of the En-positive cells of the VNC. At which step in neurogenesis do mid/H15 facilitate the formation of En expressing neurons? Since mid and H15 are not required for the formation of En-positive NBs, they may act in NBs to control lineage elaboration. Alternatively, mid/H15 may act in post-mitotic cells to maintain the expression of En. These possibilities are not mutually exclusive and require further investigation (Buescher, 2006).

This study has shown that mid and H15 are expressed in the NE from which the NBs of the rows 6, 7 and 1/2 arise, and this expression is maintained in a small subset of NBs and some of their putative progeny. Like many other segment polarity genes, mid has a dual function in the NE: it is required for NB formation within its expression domain (the anterior-most part of the segment). In addition, mid contributes to the specification of row 1/2 NBs by regulating aspects of the gene expression profile characteristic for this row. Analysis of H15 single and H15/mid-deficient embryos indicates that H15 plays no role in these processes although it has some functional activity in later aspects of neurogenesis and other developmental processes. However, even in those instances where H15 is functionally active, it plays a minor role which is only revealed in the absence of mid. For example, loss of mid function alone results in some ectopic expression of Eagle while loss of H15 alone does not. However, concomitant loss of both copies of mid and H15 results in an increase of ectopic Eagle. The differential effect on Eg expression is not due to different expression levels of mid and H15 which are approximately equal in NBs and must reflect the different intrinsic properties of both proteins although the molecular basis of these differences in protein activity is unknown. The lack of any function of H15 in the formation and early specification of NBs is most probably due to the combination of low H15 protein activity and the low level of H15 expression prior to stage 9 (Buescher, 2006).

The studies revealed that the function of mid in NB formation and specification is required predominantly in odd-numbered abdominal segments. Thus, it is likely that additional factor(s) exist that play a similar role, predominantly in even-numbered segments. The Drosophila genome was examined for additional T-box proteins with neuroectodermal expression patterns. The Drosophila genome contains eight known genes which encode T-box proteins: omb, byn, org-1, Doc1, Doc2, Doc3, mid and H15. However, besides mid and H15, none of these are neuroectodermally expressed, suggesting that the factor(s) required in even-numbered segments may not be T-box proteins. As increasingly more expression profiles of individual genes become available, suitable candidates should become obvious (Buescher, 2006).

The Tbx20 homologs midline and H15 specify ventral fate in the Drosophila melanogaster leg

Regional fates in the developing limbs of Drosophila melanogaster are controlled by selector gene transcription factors. Ventral fate in the fly leg is specified by the expression of the ligand Wingless. Evidence is presented that midline and H15, members of the Tbx20 class of T-box transcription factors, are key mediators of the Wingless signal in the formation of the ventral region of the fly leg. midline and H15 are restricted to identical ventral domains of expression through activation by Wingless and repression by the dorsal signal Decapentaplegic. midline and H15 function redundantly and cell autonomously in the formation of ventral-specific structures. Conversely, midline is sufficient to induce ventral fate. Finally, the induction of ectopic ventral fate by mid is compromised when Wingless signaling is attenuated, suggesting that Wingless acts both upstream and in parallel with midline/H15 to specify ventral fate. Based on these results, it is proposed that midline and H15 may be considered as the selector genes for ventral leg fate (Svendsen, 2009).

The Wg-dependent domain is best delineated in the second leg tarsus, where eight rows of bristles are organized around the circumference and run the length of all five tarsal segments. Wg is secreted from a stripe of cells between the primordia of the two ventral-most rows of bristles (1 and 8), which are distinct from more dorsal rows because they are peg-shaped instead of rapier-shaped. The Wg morphogen diffuses to pattern a wedge of the imaginal disc that is broader than and centered on the wg expression domain. In wg hypomorphic mutants, rows 1 and 8 are replaced with a mirror image duplication of dorsal rows 3 through to 6, resulting in a leg with double dorsal symmetry. Similar transformations are observed in clones of cells blocked for Wg signaling, where the row 1/8 bristles are transformed to rapier-shape. Other prominent Wg-dependent ventral structures include the apical bristle (AB) of the distal ventral tibia in the second leg and the ventral transverse rows (TRs) and sex combs (SCs) of the first leg (Svendsen, 2009).

The Tbx20 homologs mid and H15 are essential for the proper development of the Wg-dependent structures in the leg. In the imaginal discs, mid and H15 are expressed in identical ventral domains that are broader than and centered on the Wg domain. In the tarsus, the mid H15 domain is similar to the Wg-dependent domain, encompassing row 1 and 8 bristles and extending to, but not including, rows 2 and 7, as determined by co-staining with an antibody to Achaete, a bristle row marker. Both mid and H15 are activated in ventral cells by Wg and restricted from dorsal cells by the dorsal morphogen Dpp, but neither H15 nor mid alone is essential for leg development. However, loss of both mid and H15 in marked clones caused the autonomous transformation of the Wg-dependent peg-shaped row 1/8 bristles into lateral or dorsal rapier-like bristles. In one sample, 54 out of 56 clones transformed bristles in row 1 or 8. Similar cell-autonomous transformations were observed in the second leg tibia, in which the ventral AB was lost in mid H15 clones that span the distal tibia of the second leg. In 24 out of 26 such clones, a large bristle similar to the dorsally located pre-apical bristle (PAB) developed in place of the AB. The AB is associated with a cluster of peg-shaped bristles called spur bristles (SBs), which, like the row 1/8 bristles, were autonomously transformed to dorsal-like rapier-shaped bristles in mid H15 clones. The SCs and TRs of the first leg were also deleted in mid H15 clones. Other ventral structures were either lost or disorganized within mid H15 clones. Clones located outside the mid H15 expression domain were normal and the few ventral clones with no phenotype were small and located in structures that have no obvious D/V differences (Svendsen, 2009).

The effects of wg mutants and clones of cells unable to detect the Wg signal differ from the effects of mid H15 clones, because they also cause non-autonomous effects such as axis bifurcation or ectopic bristle rows. The axis bifurcation caused by loss of Wg function is due to ectopic dpp expression. However, neither dpp-lacZ nor the dorsal marker omb-lacZ were increased in mid H15 clones located in ventral anterior cells. The ventral-to-dorsal transformation in mid H15 clones is also not a result of a decrease in the expression of Wg, which was unchanged in ventral mid H15 clones. The homeotic gene Sex combs reduced (Scr), which is required for the development of sex combs and TRs, is expressed at high levels in the anterior tibia and basitarsus segments. mid H15 mutant clones in ventral, but not lateral or dorsal, positions downregulate Scr to background anterior levels. Taken together, these results indicate that mid and H15 are required for the specification of ventral fate downstream of Wg and for some ventral gene expression. However, mid and H15 are not required to repress dorsal gene expression (Svendsen, 2009).

Ectopic expression of mid is sufficient to induce ectopic Wg-dependent ventral structures. Since flies with H15 deleted have normal ventral patterning, mid can mediate the function of both genes. Expression of mid in the dorsal omb (bi - FlyBase) domain results in ectopic SCs and TRs in the dorsal basitarsus and distal tibia of all male first legs. This was accompanied by the ectopic expression of Scr in the omb domain, which was appropriately restricted in the P/D axis to the basitarsus and tibia. In the second leg, ectopic expression of mid in the dorsal tibia under the control of the omb-GAL4 or in small clones of mid-expressing cells result in ectopic bristles similar to the AB and SBs. Small clones of mid-expressing cells either in or adjacent to rows 2/7 and 3/6 induce ventral row 1/8 bristles cell autonomously. Similar results were seen using other GAL4 drivers expressed in the tarsus (Svendsen, 2009).

The regions of the leg where mid induces ectopic ventral structures are within the range of the ventral Wg signal, which reaches many dorsal and lateral cells to induce P/D genes such as Dll. This leaves open the possibility that Wg might act both upstream of and in parallel with mid to specify ventral fate. To test the requirement for Wg, clones of cells were generated that were compromised for Wg signaling. Mouse Lef1, which acts as a dominant negative in Wg signaling in Drosophila, was expressed, and its effects on ventral development with and without the expression of ectopic mid were compared. The clones were induced in third instar larvae, at 84 to 108 hours, when the P/D axis is independent of Wg but Wg signaling is still necessary for specifying ventral fate. mid-expressing clones induced at earlier stages can cause more extensive repatterning, with the occasional repression of dpp and non-autonomous induction of wg. Lef1 clones were distributed evenly in the dorsal, ventrolateral, dorsolateral and ventral regions of the tarsus. As expected, dorsal clones were normal and clones in the ventral-most rows often showed transformation towards more dorsal fates (9/26). Clones expressing mid were recovered much more frequently in ventral regions, suggesting that dorsal mid-expressing clones either sort to more ventral positions or they are lost. Ventrolateral or dorsolateral mid-expressing clones are often transformed to ventral character. By contrast, clones expressing both mid and Lef1 are recovered more often in lateral and dorsal cells, indicating that the sorting behavior of mid-expressing clones depends on the transduction of the Wg signal. Dorsolateral clones expressing both mid and Lef1 do not transform towards ventral fate, whereas ventrolateral clones are still sometimes transformed to ventral fate. This is consistent with a requirement for Wg in ectopic ventral development, since the dorsolateral row 3/6 bristles are further from the source of Wg signal and would be expected to be more sensitive to the effects of Lef1. A similar effect on Scr was observed, where UAS-Lef1 blocked Scr expression; this was not rescued by the simultaneous expression of UAS-mid. These results suggest that mid regulates ventral fate and Scr expression in conjunction with Wg (Svendsen, 2009).

These results suggest that the ventral expression of mid and H15 represents a major function downstream of Wg and Dpp in the D/V fate decision. The cell-autonomous requirement for mid and H15 and the ability of ectopic mid expression to induce ventral fate and gene expression in dorsal cells mean that mid and H15 meet the criteria to be defined as selector genes. In the absence of mid and H15, ventral structures may assume a dorsal fate due to the low levels of Dpp signaling found in the ventral leg. However, it is not likely that dorsal is the default fate in the leg, as lateral structures prevail when the expression of both wg and dpp is greatly reduced. Ventral fate also requires Wg signaling, suggesting that mid and H15 act to provide a molecular context for the upstream Wg morphogen to direct ventral-specific patterns of gene expression, as has been observed for other selector genes. The ventral-specific expression of mid, H15 and wg is conserved throughout several arthropod orders, suggesting that it represents a fundamental mechanism in limb patterning (Svendsen, 2009).

Transcriptional regulation of Drosophila gonad formation

The formation of the Drosophila embryonic gonad, involving the fusion of clusters of somatic gonadal precursor cells (SGPs) and their ensheathment of germ cells, provides a simple and genetically tractable model for the interplay between cells during organ formation. In a screen for mutants affecting gonad formation a SGP cell autonomous role was identified for Midline (Mid) and Longitudinals lacking (Lola). These transcription factors are required for multiple aspects of SGP behaviour including SGP cluster fusion, germ cell ensheathment and gonad compaction. The lola locus encodes more than 25 differentially spliced isoforms, and an isoform specific requirement was identified for lola in the gonad, that is distinct from that in nervous system development. Mid and Lola work in parallel in gonad formation and surprisingly Mid overexpression in a lola background leads to additional SGPs at the expense of fat body cells. These findings support the idea that although the transcription factors required by SGPs can ostensibly be assigned to those being required for either SGP specification or behaviour, they can also interact to impinge on both processes (Tripathy, 2014).

Embryonic gonad formation involves a complex interplay between two cell types and is a good model system for studying changes in cellular behaviors and cell-cell interactions, required for organogenesis. This study has identified a role for two transcription factors, Lola and Mid, in gonad development. A previous study had also identified a role for Lola in gonad formation (Weyers, 2011), and this work extends this finding in several respects. First, lola-R was identified as a specific isoform that is required by the gonad during its development. Second, Lola-R was shown to be expressed by the SGPs, and mesodermal expression of this isoform can rescue the gonad formation defects of lola null embryos. This indicates that Lola-R is required mesodermally, and that this lola isoform is sufficient to provide all Lola function in the gonad. Another zinc finger containing Lola isoform was unable to rescue the gonad defects of lola mutant embryos indicating functional differences in the distinct Lola isoforms. Whether Lola is required in the SGPs or the surrounding mesodermal cells remains an open question. It remains possible that Lola has cell autonomous functions in the SGPs as well as non-cell autonomous functions in the mesoderm, such as repressing mid or Mid function (Tripathy, 2014).

Although germ cell lola-R is not required for germ cell migration or gonad formation during embryogenesis and the Lola-R protein cannot be detected in these cells; other Lola isoforms are expressed and required in adult germ cells. In testes, lola-B and lola-I are required cell autonomously for germline stem cell maintenance and differentiation (Davies, 2013). In ovaries Lola-I is required for programmed cell death of late stage nurse cells (Bass, 2007). However, this requirement for Lola during oogenesis blocks the production of eggs from germ line clones of lola null alleles, which prevents testing whether other Lola isoforms play a role in embryonic germ cells (Tripathy, 2014).

In addition to being required for gonad formation, lola is required in the CNS. Mutants for lola null alleles show disrupted axonal tracts, however mutants in lola-R have wild-type axonal tracts. This reiterates the isoform-specific function of lola and demonstrates the ability to genetically uncouple the role of Lola in nervous system and gonad development. The lethality of flies containing the lola-R specific mutation in trans to a lola null indicates that Lola-R is also required in tissues other than the gonad, as defects in the latter would not be expected to lead to lethality (Tripathy, 2014).

The second transcription factor identified in this study was mid. Mid is a T box containing transcription factor of the tbx20 subclass with roles in embryonic patterning and axonal pathfinding. This study shows that mid mutants also have defects in gonad formation and that mid is required tissue autonomously by the SGPs (Tripathy, 2014).

To search for targets downstream of Mid and Lola in the gonad the expression was tested of genes either already identified as being important for SGP behaviour or known downstream targets in other tissues. Mid and Lola are both reported to be upstream of the cell surface receptor Robo, in the CNS. A Mid consensus binding site in the promoter region of Robo was identified with demonstrated Mid binding by chromatin immunoprecipitation (Liu, 2009). However, in the gonad of both mid and lola mutants Robo expression appeared normal. Furthermore no observable differences were found in Robo levels in the CNS of mid[B23] or mid[1] mutants compared to their heterozygous siblings in the same embryo collection. Moreover, a recently published study questioned the binding site proposed by Liu (2009) and identified a Mid consensus motif closer to the that of its vertebrate homologue, Tbx20 (Najand, 2012). Thus, although Robo is clearly required for gonad formation, whether it is downstream of Mid remains a matter of controversy (Tripathy, 2014).

Besides having demonstrated the role of two genes in gonad formation, this study has further built upon the transcriptional regulatory map in this tissue. The early SGP expression of Traffic jam (Tj) was identified as being Mid-dependent. Although Tin was not detected in late SGPs, this expression was not dependent on Mid. However, the loss of Mid and Tj expression in tin mutant SGPs, revealed a cascade of transcription factors functioning in a hierarchical and stage dependent fashion. Although, a reciprocal relationship exists between Mid and Tin in the heart, the current data demonstrates how tissues derived from the same germ layer can have different regulatory networks between the same genes (Tripathy, 2014).

Since Lola and Mid are both transcription factors, they could potentially regulate a common pool of downstream targets. Mesodermal expression of Lola-R-GFP in a mid mutant background did not rescue the mid mutant phenotype. This indicates that lola is not the sole downstream target of mid in the gonad. However, Mid over-expression in a lola mutant background results in a 'super-elongated' gonad consisting of supernumerary SGPs that span several parasegments even at late embryonic stages. This 'super-elongated' gonad results from additional SGPs being specified at the expense of fat body cells, and mirrors the effect of overexpression of the homeobox containing transcription factor Abd-A (Boyle and DiNardo, 1995 and Greig and Akam, 1995). This data raises the possibility that Abd-A balances the relative expression of Mid and Lola and suggests that the number of direct Abd-A targets is rather limited as its over-expression phenotype can be recapitulated by affecting their expression (Tripathy, 2014).

Given that Mid and Lola do not contain homeoboxes and are not required for SGP specification or maintenance, the ‘super-elongated’ phenotype seen upon over-expression of Mid in a lola background is surprising. These data argue that Lola functions to oppose Mid. Thus in the presence of wild-type Lola, overexpression of Mid does not affect SGP specification, however, in the absence of Lola, Mid overexpression results in additional SGPs (Tripathy, 2014).

A similar situation, of cell fate changes requiring shifts in expression of multiple transcription factors, occurs in the Drosophila heart. Heart cell specification requires Nkx (tin), GATA (pannier) and T box (mid, or Dorsocross) transcription factors. Whilst mis-expression of each factor alone is not sufficient to induce extra cardiac cells, combinations of these transcription factors (for example over-expression of Doc2 and pnr) can induce numbers of extra cardiac cells (Tripathy, 2014).

The results suggest that although the transcription factors required by SGPs can ostensibly be assigned to those being required for either SGP specification (such as Tin, Abd-A, Abd-B and Zfh-1) or behaviour (including D-Six4, Tj, Mid and Lola), such transcription factors can also interact to impinge on both processes. Investigating the downstream targets of Mid and Lola will provide new players and clues into how SGPs are specified and then programmed to interact with germ cells and each other to form a functional gonad (Tripathy, 2014).


H15 and leg development in insect species

Leg development in Drosophila has been studied in much detail. However, Drosophila limbs form in the larva as imaginal discs and not during embryogenesis as in most other arthropods. Appendage genes have been analyzed in the spider Cupiennius salei and the beetle Tribolium castaneum. Differences in decapentaplegic expression suggest a different mode of distal morphogen signaling suitable for the specific geometry of growing limb buds. Also, expression of the proximal genes homothorax and extradenticle (exd) is significantly altered: in the spider, exd is restricted to the proximal leg and hth expression extends distally, while in insects, exd is expressed in the entire leg and hth is restricted to proximal parts. This reversal of spatial specificity demonstrates an evolutionary shift, which is nevertheless compatible with a conserved role for this gene pair as instructor of proximal fate. Different expression dynamics of dachshund and Distal-less point to modifications in the regulation of the leg gap gene system. The significance of this finding is discussed in terms of attempts to homologize leg segments in different arthropod classes. Comparison of the expression profiles of H15 and optomotor-blind to the Drosophila patterns suggests modifications also in the dorsal-ventral patterning system of the legs. Together, these results suggest alterations in many components of the leg developmental system, namely proximal-distal and dorsal-ventral patterning, and leg segmentation. Thus, the leg developmental system exhibits a propensity to evolutionary change, which probably forms the basis for the impressive diversity of arthropod leg morphologies (Prpic, 2003).

The Drosophila genes wingless and decapentaplegic comprise the top level of a hierarchical gene cascade involved in proximal-distal (PD) patterning of the legs. It remains unclear, whether this cascade is common to the appendages of all arthropods. Here, wg and dpp are studied in the millipede Glomeris marginata, a representative of the Myriapoda. Glomeris wg (Gm-wg) is expressed along the ventral side of the appendages compatible with functioning during the patterning of both the PD and dorsal-ventral (DV) axes. Gm-wg may also be involved in sensory organ formation in the gnathal appendages by inducing the expression of Distal-less (Dll) and H15 in the organ primordia. Expression of Glomeris dpp (Gm-dpp) is found at the tip of the trunk legs as well as weakly along the dorsal side of the legs in early stages. Taking data from other arthropods into account, these results may be interpreted in favor of a conserved mode of WG/DPP signaling. Apart from the main PD axis, many arthropod appendages have additional branches (e.g., endites). It is debated whether these extra branches develop their PD axis via the same mechanism as the main PD axis, or whether branch-specific mechanisms exist. Gene expression in possible endite homologs in Glomeris argues for the latter alternative. All available data argue in favor of a conserved role of WG/DPP morphogen gradients in guiding the development of the main PD axis. Additional branches in multibranched (multiramous) appendage types apparently do not utilize the WG/DPP signaling system for their PD development. This further supports recent work on crustaceans and insects, that lead to similar conclusions (Prpic, 2004).

In Drosophila, the T-box genes optomotor-blind (omb) and H15 have been implicated in specifying the development of the dorso-ventral (DV) axis of the appendages. Results from the spider Cupiennius salei have suggested that this DV patterning system may be at least partially conserved. This study extends the study of the DV patterning genes omb and H15 to a representative of the Myriapoda in order to add to the existing comparative data set and to gain further insight into the evolution of the DV patterning system in arthropod appendages. The omb gene of the millipede Glomeris marginata is expressed on the dorsal side of all appendages including trunk legs, maxillae, mandibles, and antennae. This is similar to what is known from Drosophila and Cupiennius and suggests that the role of omb in instructing dorsal fates is conserved in arthropods. Interestingly, the lobe-shaped portions of the mouthparts do not express omb, indicating that these are ventral components and thus may be homologous to the endites present in the corresponding appendages in insects. Concerning the H15 gene, two paralogous genes were identified in Glomeris. Both genes are expressed in the sensory organs of the maxilla and antenna, but only Gm-H15-1 is expressed along the ventral side of the trunk legs. The expression is more extensive than in Cupiennius, but less so than in Drosophila. In addition, no ventral expression domain is present in the maxilla, mandible, and antenna. Because of this, the role of H15 in the determination of ventral fate remains unclear (Prpic, 2005).

The NK-2 class homeodomain factor CEH-51 and the T-box factor TBX-35 have overlapping function in C. elegans mesoderm development

The C. elegans MS blastomere, born at the 7-cell stage of embryogenesis, generates primarily mesodermal cell types, including pharynx cells, body muscles and coelomocytes. A presumptive null mutation in the T-box factor gene tbx-35, a target of the MED-1 and MED-2 divergent GATA factors, was previously found to result in a profound decrease in the production of MS-derived tissues, although the tbx-35- embryonic arrest phenotype was variable. The NK-2 class homeobox gene ceh-51 is a direct target of TBX-35 and at least one other factor, and CEH-51 and TBX-35 share functions. Embryos homozygous for a ceh-51 null mutation arrest as larvae with pharynx and muscle defects, although these tissues appear to be specified correctly. Loss of tbx-35 and ceh-51 together results in a synergistic phenotype resembling loss of med-1 and med-2. Overexpression of ceh-51 causes embryonic arrest and generation of ectopic body muscle and coelomocytes. These data show that TBX-35 and CEH-51 have overlapping function in MS lineage development. As T-box regulators and NK-2 homeodomain factors are both important for heart development in Drosophila and vertebrates, these results suggest that these regulators function in a similar manner in C. elegans to specify a major precursor of mesoderm (Broitman-Maduro, 2009).

Zebrafish Tbx20 homolog and heart development

T-box transcription factors are critical regulators of early embryonic development. A novel zebrafish T-box transcription factor, hrT (H15-related T box) has been characterized that is a close relative of Drosophila H15 and a recently identified human gene. Drosophila H15 and zebrafish hrT are both expressed early during heart formation, in strong support of previous work postulating that vertebrate and arthropod hearts are homologous structures with conserved regulatory mechanisms. The timing and regulation of zebrafish hrT expression in anterior lateral plate mesoderm suggest a very early role for hrT in the differentiation of the cardiac precursors. hrT is coexpressed with gata4 and nkx2.5 not only in anterior lateral plate mesoderm but also in noncardiac mesoderm adjacent to the tail bud, suggesting that a conserved regulatory pathway links expression of these three genes in cardiac and noncardiac tissues. Finally, hrT expression was examined in pandora mutant embryos, since these have defects in many of the tissues that express hrT, including the heart. hrT expression is much reduced in the early heart fields of pandora mutants, whereas it is ectopically expressed subsequently. Using hrT expression as a marker, a midline patterning defect is described in pandora affecting the anterior hindbrain and associated midline mesendodermal derivatives. The possibility that the cardiac ventricular defect previously described in pandora and the midline defects described in this study are related (Griffin, 2000).

The T-box genes constitute a family of transcriptional regulator genes that have been implicated in a variety of developmental processes ranging from the formation of germ layers to the regionalization of the central nervous system. The cloning and expression pattern of a new T-box gene from zebrafish, which was named tbx20, is described. tbx20 is an ortholog of two other T-box genes isolated from animals of different phyla - H15 of Drosophila melanogaster and tbx-12 of Caenorhabditis elegans, suggesting that the evolutionary origin of this gene predates the divergence between the protostomes and deuterostomes. During development, tbx20 is expressed in embryonic structures of both mesodermal and ectodermal origins, including the heart, cranial motor neurons, and the roof of the dorsal aorta (Ahn, 2000).

The recently identified zebrafish T-box gene hrT is expressed in the developing heart and in the endothelial cells forming the dorsal aorta. Orthologs of hrT are expressed in cardiovascular cells from Drosophila to mouse, suggesting that the function of hrT is evolutionarily conserved. The role of hrT in cardiovascular development, however, has not thus far been determined in any animal model. Using morpholino antisense oligonucleotides, it was shown that zebrafish embryos lacking hrT function have dysmorphic hearts and an absence of blood circulation. Although the early events in heart formation are normal in hrT morphant embryos, subsequently the hearts fail to undergo looping, and late onset defects in chamber morphology and gene expression are observed. In particular, it was found that the loss of hrT function leads to a dramatic upregulation of tbx5, a gene required for normal heart morphogenesis. Conversely, overexpression of hrT is shown to cause a significant downregulation of tbx5, indicating that one key role of hrT is to regulate the levels of tbx5. It was also found that HrT is required to inhibit the expression of the blood lineage markers gata1 and gata2 in the most posterior lateral plate mesoderm. Finally, it has been shown that HrT is required for vasculogenesis in the trunk, leading to similar vascular defects to those observed in midline mutants such as floating head. hrT expression in the vascular progenitors depends upon midline mesoderm, indicating that this expression is one important component of the response to a midline-derived signal during vascular morphogenesis (Szeto, 2002).

Mutations in T-box genes are the cause of several congenital diseases and are implicated in cancer. Tbx20-null mice exhibit severely hypoplastic hearts and express Tbx2, which is normally restricted to outflow tract and atrioventricular canal, throughout the heart. Tbx20 mutant hearts closely resemble those seen in mice overexpressing Tbx2 in myocardium, suggesting that upregulation of Tbx2 can largely account for the cardiac phenotype in Tbx20-null mice. Evidence is provided that Tbx2 is a direct target for repression by Tbx20 in developing heart. Tbx2 directly binds to the Nmyc1 promoter in developing heart and can repress expression of the Nmyc1 promoter in transient transfection studies. Repression of Nmyc1 (N-myc) by aberrantly regulated Tbx2 can account in part for the observed cardiac hypoplasia in Tbx20 mutants. Nmyc1 is required for growth and development of multiple organs, including the heart, and overexpression of Nmyc1 is associated with childhood tumors. Despite its clinical relevance, the factors that regulate Nmyc1 expression during development are unknown. These data present a paradigm by which T-box proteins regulate regional differences in Nmyc1 expression and proliferation to effect organ morphogenesis. A model is presented whereby Tbx2 directly represses Nmyc1 in outflow tract and atrioventricular canal of the developing heart, resulting in relatively low proliferation. In chamber myocardium, Tbx20 represses Tbx2, preventing repression of Nmyc1 and resulting in relatively high proliferation. In addition to its role in regulating regional proliferation, Tbx20 regulates expression of a number of genes that specify regional identity within the heart, thereby coordinating these two important aspects of organ development (Cai, 2005).

To elucidate the function of the T-box transcription factor Tbx20 in mammalian development, a graded loss-of-function series was generated by transgenic RNA interference in entirely embryonic stem cell-derived mouse embryos. Complete Tbx20 knockdown results in defects in heart formation, including hypoplasia of the outflow tract and right ventricle, which derive from the anterior heart field (AHF), and decrease in the expression of Nkx2-5 and Mef2c, transcription factors required for AHF formation. A mild knockdown led to persistent truncus arteriosus (unseptated outflow tract) and hypoplastic right ventricle, entities similar to human congenital heart defects; this demonstrates a critical requirement for Tbx20 in valve formation. Finally, an intermediate knockdown revealed a role for Tbx20 in motoneuron development, specifically in the regulation of the transcription factors Isl2 and Hb9, which are important for terminal differentiation of motoneurons. Tbx20 can activate promoters/enhancers of several genes in cultured cells, including the Mef2c AHF enhancer and the Nkx2-5 cardiac enhancer. The Mef2c AHF enhancer relies on Isl1- and Gata-binding sites. A similar Isl1 binding site has been identified in the Nkx2-5 AHF enhancer, which in transgenic mouse embryos is essential for activity in a large part of the heart, including the outflow tract. Tbx20 synergizes with Isl1 and Gata4 to activate both the Mef2c and Nkx2-5 enhancers, thus providing a unifying mechanism for gene activation by Tbx20 in the AHF. It is thus concluded that Tbx20 is positioned at a critical node in transcription factor networks required for heart and motoneuron development where it dose-dependently regulates gene expression (Takeuchi, 2005).

The genetic hierarchies guiding lineage specification and morphogenesis of the mammalian embryonic heart are poorly understood. It has been shown by gene targeting that murine T-box transcription factor Tbx20 plays a central role in these pathways, and has important activities in both cardiac development and adult function. Loss of Tbx20 results in death of embryos at mid-gestation with grossly abnormal heart morphogenesis. Underlying these disturbances is a severely compromised cardiac transcriptional program, defects in the molecular pre-pattern, reduced expansion of cardiac progenitors and a block to chamber differentiation. Notably, Tbx20-null embryos show ectopic activation of Tbx2 across the whole heart myogenic field. Tbx2 encodes a transcriptional repressor normally expressed in non-chamber myocardium, and in the atrioventricular canal it has been proposed to inhibit chamber-specific gene expression through competition with positive factor Tbx5. These data demonstrate a repressive activity for Tbx20 and place it upstream of Tbx2 in the cardiac genetic program. Thus, hierarchical, repressive interactions between Tbx20 and other T-box genes and factors underlie the primary lineage split into chamber and non-chamber myocardium in the forming heart, an early event upon which all subsequent morphogenesis depends. Additional roles for Tbx20 in adult heart integrity and contractile function were revealed by in-vivo cardiac functional analysis of Tbx20 heterozygous mutant mice. These data suggest that mutations in human cardiac transcription factor genes, possibly including TBX20, underlie both congenital heart disease and adult cardiomyopathies (Stennard, 2005).

Tbx20, a member of the T-box family of transcriptional regulators, shows evolutionary conserved expression in the developing heart. In the mouse, Tbx20 is expressed in the cardiac crescent, then in the endocardium and myocardium of the linear and looped heart tube before it is restricted to the atrioventricular canal and outflow tract in the multi-chambered heart. Tbx20 is required for progression from the linear heart tube to a multi-chambered heart. Mice carrying a targeted mutation of Tbx20 show early embryonic lethality due to hemodynamic failure. A linear heart tube with normal anteroposterior patterning is established in the mutant. The tube does not elongate, indicating a defect in recruitment of mesenchyme from the secondary heart field, even though markers of the secondary heart field are not affected. Furthermore, dorsoventral patterning of the tube, formation of working myocardium, looping, and further differentiation and morphogenesis fail. Instead, Tbx2, Bmp2 and vinexin alpha (Sh3d4), genes normally restricted to regions of primary myocardium and lining endocardium, are ectopically expressed in the linear heart tube of Tbx20 mutant embryos. Because Tbx2 is both necessary and sufficient to repress chamber differentiation Tbx20 may ensure progression to a multi-chambered heart by repressing Tbx2 in the myocardial precursor cells of the linear heart tube destined to form the chambers (Singh, 2005).

Characterization of Xenopus Tbx20 homolog

The regulation of vertebrate eye development requires the activity of many transcription factors. The T-box factor Tbx12 is necessary for normal development of the retina. Tbx12 is expressed during early stages of retinal development in multiple species of vertebrate embryos. mRNAs encoding wild type and mutant forms of Tbx12 were injected into Xenopus embryos. The Tbx12 injected embryos exhibit multiple defects in eye development including reduced eye size and disruption of normal retinal laminar organization. Tbx12 appears to function as a repressor of transcription during eye development. These results indicate that Tbx12 activity is required for the proper generation and organization of retinal cells in the vertebrate eye (Carson, 2004).

Chick Tbx20 and heart development

Little is known about the molecular mechanisms involved with the initial specifications of the cardiac mesoderm. In order to identify potential regulatory factors that play important roles in early heart specification, the chick H15-related T-box gene was isolated and its expression pattern was analzyed during early development. The chick Tbx20 gene was found to be highly homologous to human, mouse, and zebrafish hrT/Tbx20. Its expression is initially detected in the posterior lateral mesoderm, after which it expands to the anterior and is intensively co-expressed with a cardiogenic gene, Nkx2.5, in the anterior lateral mesoderm (Iio, 2001).

Extensive misexpression studies were carried out to explore the roles played by Tbx5, the expression of which is excluded from the right ventricle (RV) during cardiogenesis. When Tbx5 is misexpressed ubiquitously, ventricular septum is not formed, resulting in a single ventricle. In such a heart, left ventricle (LV)-specific ANF gene is induced. In search of the putative RV factor(s), chick Tbx20 was found to be expressed in the RV, showing a complementary fashion to Tbx5. In the Tbx5-misexpressed heart, this gene is repressed. When misexpression is spatially partial, leaving small Tbx5-negative area in the right ventricle, ventricular septum is shifted rightwards, resulting in a small RV with an enlarged LV. Focal expression induces an ectopic boundary of Tbx5-positive and -negative regions in the right ventricle, at which an additional septum is formed. Similar results were obtained from the transient transgenic mice. In such hearts, expression patterns of dHAND and eHAND are changed with definitive cardiac abnormalities. Furthermore, human ANF promoter is synergistically activated by Tbx5, Nkx2.5 and GATA4. This activation is abrogated by Tbx20, implicating the pivotal roles of interactions among these heart-specific factors. Taken together, these data indicate that Tbx5 specifies the identity of LV through tight interactions among several heart-specific factors, and highlight the essential roles of Tbx5 in cardiac development (Takeuchi, 2003).

Identification and characterization of mammalian Tbx20

T-box transcription factors contain a novel type of DNA-binding domain, the T-box domain, and are encoded by an ancient gene family. Four T-box genes, omb, Trg, org-1, and H15, have been identified in Drosophila, whereas in mammals the T-box gene family has expanded, and 12 human T-box genes have been isolated. A new human T-box gene, TBX20, and its mouse homologue Tbx20, have been identified that are more closely related to the Drosophila H15 gene than to any known vertebrate gene. H15 expression in leg imaginal discs correlates with commitment to a ventral fate, implicating this gene in early patterning events. TBX20 is expressed in the fetal heart, eye, and limb, and during embryogenesis in the mouse, Tbx20 is expressed in the developing heart, eye, ventral neural tube, and limbs, indicating a possible role in regulating development of these tissues. The TBX20 gene maps to chromosome 7p14-p15. An association between TBX20 and loci for retinitis pigmentosa, RP9, and blepharophimosis syndrome, BPES, have been excluded (Meins, 2000).

T-box genes encode transcription factors that regulate many developmental processes. A novel mouse T-box gene, Tbx12, has been cloned. Tbx12 is the vertebrate homologue of the Drosophila H15 gene and the Caenorhabditis elegans tbx-12 gene. Tbx12 is expressed in extraembryonic tissues such as the amnion and allantois. In the embryo, Tbx12 is strongly expressed in the neural retina and the heart (Carson, 2000).

T-box genes constitute a conserved multi-gene family with important roles in many developmental processes. The cloning and expression analysis is described of a novel mouse T-box gene, Tbx20. Expression is prominent in the extraembryonic mesoderm, in the developing heart, the eye anlage and motor neurons of hindbrain and spinal cord (Kraus, 2001).

Mammalian Tbx20 and heart development

Tbx20 is a member of the T-box transcription factor family expressed in the forming hearts of vertebrate and invertebrate embryos. This study reports analysis of Tbx20 expression during murine cardiac development and assessment of DNA-binding and transcriptional properties of Tbx20 isoforms. Tbx20 was expressed in myocardium and endocardium, including high levels in endocardial cushions. cDNAs generated by alternative splicing encode at least four Tbx20 isoforms, and Tbx20a uniquely carries strong transactivation and transrepression domains in its C terminus. Isoforms with an intact T-box bind specifically to DNA sites resembling the consensus brachyury half site, although with less avidity compared with the related factor, Tbx5. Tbx20 physically interacts with cardiac transcription factors Nkx2-5, GATA4, and GATA5, collaborating to synergistically activate cardiac gene expression. Among cardiac GATA factors, there was preferential synergy with GATA5, implicated in endocardial differentiation. In Xenopus embryos, enforced expression of Tbx20a, but not Tbx20b, leads to induction of mesodermal and endodermal lineage markers as well as cell migration, indicating that the long Tbx20a isoform uniquely bears functional domains that can alter gene expression and developmental behaviour in an in vivo context. It is proposed that Tbx20 plays an integrated role in the ancient myogenic program of the heart, and has been additionally coopted during evolution of vertebrates for endocardial cushion development (Stennard, 2003).

The T-box transcription factors play critical roles in embryonic development including cell type specification, tissue patterning, and morphogenesis. Several T-box genes are expressed in the heart and are regulators of cardiac development. At the earliest stages of heart development, two of these genes, Tbx5 and Tbx20, are co-expressed in the heart-forming region but then become differentially expressed as heart morphogenesis progresses. Although Tbx5 and Tbx20 belong to the same gene family and share a highly conserved DNA-binding domain, their transcriptional activities are distinct. The C-terminal region of the Tbx5 protein is a transcriptional activator, while the C terminus of Tbx20 can repress transcription. Tbx5, but not Tbx20, activates a cardiac-specific promoter (atrial natriuretic factor (ANF)) alone and synergistically with other transcription factors. In contrast, Tbx20 represses ANF promoter activity and also inhibits the activation mediated by Tbx5. Of the two T-box binding consensus sequences in the promoter of ANF, only T-box binding element 1 (TBE1) is required for the synergistic activation of ANF by Tbx5 and GATA4, but TBE2 is required for repression by Tbx20. To elucidate upstream signaling pathways that regulate Tbx5 and Tbx20 expression, recombinant bone morphogenetic protein-2 was added to cardiogenic explants from chick embryos. Using real time reverse transcription-PCR, it was demonstrated that Tbx20, but not Tbx5, is induced by bone morphogenetic protein-2. Collectively these data demonstrate clear differences in both the expression and function of two related transcription factors and suggest that the modulation of cardiac gene expression can occur as a result of combinatorial regulatory interactions of T-box proteins (Plageman, 2004).

Members of the T-box family of proteins play a fundamental role in patterning the developing vertebrate heart; however, the precise cellular requirements for any one family member and the mechanism by which individual T-box genes function remains largely unknown. In this study, the cellular and molecular relationship between two T-box genes, Tbx5 and Tbx20, was investigated. Blocking Tbx5 or Tbx20 produces phenotypes that display a high degree of similarity, as judged by overall gross morphology, molecular marker analysis and cardiac physiology, implying that the two genes are required for and have non-redundant functions in early heart development. In addition, although co-expressed, Tbx5 and Tbx20 are not dependent on the expression of one another, but rather have a synergistic role during early heart development. Consistent with this proposal, it is shown that TBX5 and TBX20 can physically interact, the interaction domains were mapped, and a cellular interaction was shown for the two proteins in cardiac development, thus providing the first evidence for direct interaction between members of the T-box gene family (Brown, 2005).

The cardiac conduction system comprises a specialized tract of electrically coupled cardiomyocytes responsible for impulse propagation through the heart. Abnormalities in cardiac conduction are responsible for numerous forms of cardiac arrhythmias, but relatively little is known about the gene regulatory mechanisms that control the formation of the conduction system. This study demonstrates that a distal enhancer for the connexin 30.2 (Cx30.2, also known as Gjd3) gene, which encodes a gap junction protein required for normal atrioventricular (AV) delay in mice, is necessary and sufficient to direct expression to the developing AV conduction system (AVCS). Moreover, this enhancer requires Tbx5 and Gata4 for proper expression in the conduction system, and Gata4+/- mice have short PR intervals indicative of accelerated AV conduction. These results implicate Gata4 in conduction system function and provide a clearer understanding of the transcriptional pathways that impact normal AV delay (Munshi, 2009).

TBX20 has been shown to be essential for vertebrate heart development. Mutations within the TBX20 coding region are associated with human congenital heart disease, and the loss of Tbx20 in a wide variety of model systems leads to cardiac defects and eventually heart failure. Despite the crucial role of TBX20 in a range of cardiac cellular processes, the signal transduction pathways that act upstream of Tbx20 remain unknown. This study identified and characterized a conserved 334 bp Tbx20 cardiac regulatory element that is directly activated by the BMP/SMAD1 signaling pathway. This element is both necessary and sufficient to drive cardiac-specific expression of Tbx20 in Xenopus, and blocking SMAD1 signaling in vivo specifically abolishes transcription of Tbx20, but not that of other cardiac factors, such as Tbx5 and MHC, in the developing heart. Activation of Tbx20 by SMAD1 is mediated by a set of novel, non-canonical, high-affinity SMAD-binding sites located within this regulatory element, and phospho-SMAD1 directly binds a non-canonical SMAD1 site in vivo. Finally, it was shown that these non-canonical sites are necessary and sufficient for Tbx20 expression in Xenopus, and that reporter constructs containing these sites are expressed in a cardiac-specific manner in zebrafish and mouse. Collectively, these findings define Tbx20 as a direct transcriptional target of the BMP/SMAD1 signaling pathway during cardiac maturation (Mandel 2010).


Search PubMed for articles about Drosophila H15 and Midline

Ahn, D. G., Ruvinsky, I., Oates, A. C., Silver, L. M. and Ho, R. K. (2000). tbx20, a new vertebrate T-box gene expressed in the cranial motor neurons and developing cardiovascular structures in zebrafish. Mech. Dev. 95(1-2): 253-8. 10906473

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Broitman-Maduro, G., et al. (2009). The NK-2 class homeodomain factor CEH-51 and the T-box factor TBX-35 have overlapping function in C. elegans mesoderm development. Development 136(16): 2735-46. PubMed Citation: 19605496

Brook, W. J., et al. (1993). Gene expression during imaginal disc regeneration detected using enhancer-sensitive P-elements. Development 117(4): 1287-97. 8404531

Brook, W. J. and Cohen, S. M. (1996). Antagonistic interactions between wingless and decapentaplegic responsible for dorsal-ventral pattern in the Drosophila Leg. Science 273(5280): 1373-7. 8703069

Brown, D. D., et al. (2005). Tbx5 and Tbx20 act synergistically to control vertebrate heart morphogenesis. Development. 132(3): 553-63. 15634698

Buescher, M., Svendsen, P. C., Tio, M., Miskolczi-McCallum, C., Tear, G., Brook, W. J. and Chia, W. (2004). Drosophila T box proteins break the symmetry of hedgehog-dependent activation of wingless. Curr. Biol. 14(19): 1694-702. 15458640

Buescher, M., Tio, M., Tear, G., Overton, P. M., Brook, W. J. and Chia, W. (2006). Functions of the segment polarity genes midline and H15 in Drosophila melanogaster neurogenesis. Dev. Biol. 292(2): 418-29. 16499900

Cai, C. L., et al. (2005). T-box genes coordinate regional rates of proliferation and regional specification during cardiogenesis. Development 132(10): 2475-87. 15843407

Carson, C. T., Kinzler, E. R. and Parr, B. A. (2000). Tbx12, a novel T-box gene, is expressed during early stages of heart and retinal development. Mech. Dev. 96(1): 137-40. 10940636

Carson, C. T., Pagratis, M. and Parr, B. A. (2004). Tbx12 regulates eye development in Xenopus embryos. Biochem. Biophys. Res. Commun. 318(2): 485-9. 15120626

Davies, E. L., Lim, J. G., Joo, W. J., Tam, C. H. and Fuller, M. T. (2013). The transcriptional regulator lola is required for stem cell maintenance and germ cell differentiation in the Drosophila testis. Dev Biol 373: 310-321. PubMed ID: 23159836

Fregoso Lomas, M., De Vito, S., Boisclair Lachance, J.F., Houde, J. and Nilson, L.A. (2016). Determination of EGFR signaling output by opposing gradients of BMP and JAK/STAT activity. Curr Biol 26(19):2572-2582. PubMed ID: 27593379

Gaziova, I. and Bhat, K. M. (2009). Ancestry-independent fate specification and plasticity in the developmental timing of a typical Drosophila neuronal lineage. Development 136(2): 263-74. PubMed Citation: 19088087

Griffin, K. J., Stoller, J., Gibson, M., Chen, S., Yelon, D., Stainier, D. Y. and Kimelman, D. (2000). A conserved role for H15-related T-box transcription factors in zebrafish and Drosophila heart formation. Dev. Biol. 218(2): 235-47. 10656766

Iio, A., Koide, M., Hidaka, K. and Morisaki, T. (2001). Expression pattern of novel chick T-box gene, Tbx20. Dev. Genes Evol. 211(11): 559-62. 11862462

Johnson, A., Bergman, C., Kreitman, M. and Newfeld, S. (2003). Embryonic enhancers in the dpp disk region regulate a second round of Dpp signaling from the dorsal ectoderm to the mesoderm that represses Zfh-1 expression in a subset of pericardial cells. Dev. Biol. 262: 137-151. PubMed citation: 14512024

Johnson, A. N., Burnett, L. A., Sellin, J., Paululat, A. and Newfeld, S. J. (2007). Defective decapentaplegic signaling results in heart overgrowth and reduced cardiac output in Drosophila. Genetics 176(3): 1609-24. PubMed citation: 17507674

Johnson, A. N., Mokalled, M. H., Haden, T. N. and Olson, E. N. (2011). JAK/Stat signaling regulates heart precursor diversification in Drosophila. Development 138(21): 4627-38. PubMed Citation: 21965617

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Kolsch, V. and Paululat, A. (2002). The highly conserved cardiogenic bHLH factor Hand is specifically expressed in circular visceral muscle progenitor cells and in all cell types of the dorsal vessel during Drosophila embryogenesis. Dev. Genes Evol. 212(10): 473-85. 12424518

Kraus, F., Haenig, B. and Kispert, A. (2001). Cloning and expression analysis of the mouse T-box gene tbx20. Mech. Dev. 100(1): 87-91. 11118890

Liu, Q. X., Hiramoto, M., Ueda, H., Gojobori, T., Hiromi, Y. and Hirose, S. (2009). Midline governs axon pathfinding by coordinating expression of two major guidance systems. Genes Dev 23: 1165-1170. PubMed ID: 19451216

Manavalan, M. A., Gaziova, I. and Bhat, K. M. (2013). The Midline protein regulates axon guidance by blocking the reiteration of neuroblast rows within the Drosophila ventral nerve cord. PLoS Genet 9: e1004050. PubMed ID: 24385932

Mandel, E. M., et al. (2010). The BMP pathway acts to directly regulate Tbx20 in the developing heart. Development 137(11): 1919-29. PubMed Citation: 20460370

Meins, M., Henderson, D. J., Bhattacharya, S. S. and Sowden, J. C. (2000). Characterization of the human TBX20 gene, a new member of the T-Box gene family closely related to the Drosophila H15 gene. Genomics 67(3): 317-32. 10936053

Miskolczi-McCallum, C. M., Scavetta, R. J., Svendsen, P. C., Soanes, K. H. and Brook, W. J. (2005). The Drosophila melanogaster T-box genes midline and H15 are conserved regulators of heart development. Dev. Biol. 2005 278(2): 459-72. 15680363

Munshi, N. V., et al. (2009). Cx30.2 enhancer analysis identifies Gata4 as a novel regulator of atrioventricular delay. Development. 136(15): 2665-74. PubMed Citation: 19592579

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Plageman, T. F. and Yutzey, K. E. (2004). Differential expression and function of Tbx5 and Tbx20 in cardiac development. J. Biol. Chem. 279(18): 19026-34. 14978031

Prpic, N. M., Janssen, R., Wigand, B., Klingler, M. and Damen, W. G. (2003). Gene expression in spider appendages reveals reversal of exd/hth spatial specificity, altered leg gap gene dynamics, and suggests divergent distal morphogen signaling. Dev. Biol. 264(1): 119-40. 14623236

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Singh, M. K., et al. (2005). Tbx20 is essential for cardiac chamber differentiation and repression of Tbx2. Development 132(12): 2697-707. 15901664

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Stennard, F. A., et al. (2005). Murine T-box transcription factor Tbx20 acts as a repressor during heart development, and is essential for adult heart integrity, function and adaptation. Development 132(10): 2451-62. 15843414

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Takeuchi, J. K., et al. (2005). Tbx20 dose-dependently regulates transcription factor networks required for mouse heart and motoneuron development. Development 132(10): 2463-74. 15843409

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date revised: 12 December 2016

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