BIOLOGICAL OVERVIEW

Two of the most important and better understood types of transcription factors are homeodomain proteins and zinc finger proteins. Less well characterized is a family of transcription factors possessing both zinc fingers and homeodomains. Drosophila has two zinc finger homeodomain (ZFH) proteins: ZFH-1 and ZFH-2. Both are homologous to the two known vertebrate ZFH family members. A vertebrate homolog of ZFH-1 is delta EF1, which binds to the essential element of a crystallin gene. ZFH-1 and delta EF1 each have a central homeodomain and N-terminal and C-terminal clusters of zinc fingers. A vertebrate homolog of Drosophila ZFH-2 is mouse ATBF1 encoding a 406-kDa protein containing four homeodomains and 23 zinc-finger motifs. Drosophila ZFH-2 has 3 homodomains and 17 zinc-finger domains. One is left wondering what could be the function of the multiplicity of zinc-finger domains and homeodomains in these complex proteins.

Both Drosophila ZFH proteins are expressed in the nervous system. ZFH-2 binds to a regulatory region of the DOPA decarboxylase gene (DDC). The regulatory region to which ZFH-2 binds is important for cell-specific expression of DDC in the Drosophila central nervous system (CNS). The in vivo profile of ZFH-2 in the larval CNS shows intriguing overlap with DDC in specific serotonin and dopamine neurons (Lundell, 1992). For more information about these neurons see Islet, Engrailed and Huckebein. zfh-1 is expressed in the majority of identified motor neurons of the developing CNS. Neural phenotypes for zfh-1 mutants have not been reported (Lai, 1991).

Loss of zfh-1 function results in various degrees of local errors in mesodermal cell fate or positioning. The ventral-oblique and the dorsal muscles are usually the most severely affected. There are a variety of errors in segregation of muscle precursors. Mutants have missing muscles, misplaced muscles, and nuclei within a muscle are disorganized. The foregut and hindgut appear normal, but the midgut, structured by expression of zfh-1 in the visceral mesoderm, is abnormal. Constrictions start to form but rarely complete the subdivision of the yolk, and the elongation and narrowing of the gut occurs in a partial and very uneven fashion. Other defects are seen in the heart, gonads and pole cells. Adult muscle precursors are abnormal (Lai, 1993).

Of great interest is the expression of ZFH-1 in head mesoderm. Here the gene's expression does not require twist or snail, both of which are required for visceral and somatic mesoderm expression of ZFH-1. Head mesoderm is the earliest expression domain of ZFH-1. Currently the function of ZFH-1 in this tissue is unknown. Of equal interest is the expression of ZFH-1 in motor neurons. Again, the function of neuronal expression of ZFH-1 is unknown.

Loss of zfh-1 activity disrupts the development of two distinct mesodermal populations: the caudal visceral mesoderm (along which germ cells migrate) and the gonadal mesoderm (the final destination of the germ cells). The caudal visceral mesoderm facilitates the migration of germ cells from the endoderm to the mesoderm. Zfh-1 is also expressed in the gonadal mesoderm throughout the development of this tissue. Ectopic expression of Zfh-1 is sufficient to induce additional gonadal mesodermal cells and to alter the temporal course of gene expression within these cells. Germ cell migration was also analyzed in brachyenteron mutant embryos. Like zfh-1, byn is required for the migration of the caudal visceral mesoderm, but unlike zfh-1, it is not required for gonadal mesoderm development. Since byn and zfh-1 both disrupt caudal visceral mesoderm migration and show similar defects in germ cell migration, it is proposed that in wild-type embryos, the caudal visceral mesoderm facilitates the transition of many germ cells from the endoderm to the lateral mesoderm. abdominal-A is also required for gonadal mesoderm specification. Zfh-1 expression was analyzed in abdA mutants. Zfh-1 is expressed normally in mesodermal clusters at stage 10, however, its levels are not enhanced in PS10-12 during stage ll. The loss of high Zfh-1 expression correlates with the failure of SGP specification in abdA mutants. Although abdA is required for SGP specification, the initial stages of germ cell migration are unaffected in abdA mutant embryos (Broihier, 1998).

Analysis of a tinman;zfh-1 double mutant shows that zfh-1 acts in conjunction with tinman, another homeodomain protein, in the specification of lateral mesodermal derivatives, including the gonadal mesoderm. It is unlikely, however that tin and zfh-1 fit neatly into a linear hierarchy controlling gonadal mesoderm determination. The early broad expression of tin is required for SGP development. However, zfh-1 is not required for this expression, suggesting that zfh-1 is not upstream of tin. Furthermore, since germ cell association with SGPs is blocked in zfh-1 mutants but not in tin mutants, it seems unlikely that tin acts upstream of zfh-1 in SGP development. These observations suggest that tin and zfh-1 function in parallel in gonadal mesoderm development (Broihier, 1998).

A series of inductive signals are necessary to subdivide the mesoderm in order to allow the formation of the progenitor cells of the heart. Mesoderm-endogenous transcription factors, such as those encoded by twist and tinman, seem to cooperate with these signals to confer correct context and competence for a cardiac cell fate. Additional factors are likely to be required for the appropriate specification of individual cell types within the forming heart. Similar to tinman, the zinc finger- and homeobox-containing gene zfh-1 is expressed in the early mesoderm and later in the forming heart, suggesting a possible role in heart development. zfh-1 is specifically required for formation of the even-skipped (eve)-expressing subset of pericardial cells (EPCs), without affecting the formation of their siblings, the founders of a dorsal body wall muscle (DA1). In addition to zfh-1, mesodermal eve itself appears to be needed for correct EPC differentiation, possibly as a direct target of zfh-1. Epistasis experiments show that zfh-1 specifies EPC development independent of numb, the lineage gene that controls DA1 founder versus EPC cell fate. The combinatorial control mechanisms that specify the EPC cell fate in a spatially precise pattern within the embryo are discussed (Su, 1999). zfh-1 and the components of the numb pathway are not the only factors required for specifying EPC or DA1 founder fates (or for eve expression characteristic of these fates). A transcription factor encoded by the lethal-of-scute gene is expressed in a cluster of mesodermal cells out of which the EPC and other muscle progenitors emerge aided by a laterally inhibitory mechanism. lethal-of-scute, however, as well as another transcription factor encoded by the Krüppel gene, which is expressed in the DA1 (and other muscle) founder cells, are only weakly required for the corresponding muscles to form. In contrast, the Drosophila EGF signal transduction pathway plays an essential role in DA1 specification. For example, in the absence of the secreted EGF-receptor ligand spitz, the number of EPCs is normal but nearly all the DA1 muscles fail to form. Since DA1 founders and EPCs are likely to derive from common precursors and the phenotype of spi mutants is the opposite of zfh-1, it was decided to determine whether or not zfh-1 and spitz function as part of a common genetic pathway. The phenotype of spitz;zfh-1 double mutants was examined. In these double mutants, neither EPC- nor DA1-specific eve expression is present, suggesting that the Egf-r pathway is required for DA1 differentiation independently of zfh-1. This raises the question of whether or not Egf-r pathway activation is required for providing the correct DA1 differentiation context in a way that is reminiscent of zfh-1 function, which provides a context for EPC differentiation. If yes, it would be expected that spitz, like zfh-1, functions independently of the numb pathway. Indeed, when numb is mesodermally overexpressed in spitz mutant embryos, a phenotype similar to that of spitz;zfh-1 double mutants is observed: neither EPC- and nor DA1-specific eve expression is observed. Taken together, these results suggest that correct cell type-specific differentiation depends on both asymmetric segregation of cell fate determinants during cell division as well as on the appropriate regional context. In this case, the context information (zfh-1 or Egf-r activity) does not need to be originating from a spatially localized source, but may act in concert with other mesodermal context determinants (e.g., tinman) (Su, 1999).

eve is well known for its function in ectodermal segmentation. eve also participates in the patterning of the early mesoderm. eve null mutants lack visceral and cardiac mesoderm altogether. As described above, zfh-1 is required for the formation and/or expression of eve in EPCs. Thus, mesodermal eve expression itself may be needed for correct EPC differentiation. To address this question, a temperature-sensitive allele of eve (eve^ID), which produces a non-functional but nevertheless antigenic protein at the non-permissive temperature, was used. When eve^ID mutant embryos are shifted to the non-permissive temperature for 2 hours at early stage 11, the number of EPCs (expressing eve) is drastically reduced: on average only 24% of EPCs are present as compared to wild type. DA1 muscle formation also seems to be affected, but to a lesser degree. The EPC deficiency is less severe when the temperature shifts occurs earlier or later in development. Interestingly, some of the remaining EPCs in early stage 11 shifted embryos are located at some distance from the heart tube, suggesting that eve function during this critical time period is required for correct differentiation of the EPCs. Thus, in the absence of eve, the forming EPCs lose their association with the heart and disappear. Temperature shifts during the temperature-sensitive period for EPC formation also affect the overall pericardial cell population, as seen in zfh-1 mutant embryos, perhaps due in part to the lack of EPCs. In contrast to the zfh-1 phenotype, however, the number of cardial cells, heart tube formation and overall body muscle formation are not significantly affected in early stage 11 shifted eve^ID embryos, which is not surprising since eve is not expressed in these tissues during the critical period for EPC formation. Although eve inactivation at earlier stages also perturbs neural development (that is, RP2 neurons), in addition to visceral and somatic muscle formation, stage 11 shifts show no morphologically detectable CNS defects. It has been concluded that eve, in addition to zfh-1, is required for the proper differentiation of the EPCs at the time when the eve progenitors normally appear (Su, 1999).

Since zfh-1 is required for eve expression in the forming EPCs and eve function is required for EPC differentiation, it was asked if eve function is sufficient to promote EPC development in the absence of zfh-1. Mesodermal expression of eve may be autoregulated, as is the case for the eve late stripe expression element. Thus, eve expression may need to be activated at least until autoregulation is initiated. The Gal4 system was used to ectopically express eve in the mesoderm of zfh-1 mutant embryos until (but not beyond) stage 11. At stage 14/15, EPC-specific endogenous Eve protein expression was examined. In order to achieve this, the twist promoter was used to drive Gal4 expression, which in turn drives the eve cDNA under the control of UAS Gal4-binding sites. This protocol to overexpress eve in the mesoderm of zfh-1 mutant embryos partially restores the formation of EPCs. These results support the hypothesis that eve acts downstream of zfh-1 and that it is required itself for the proper formation of EPCs. Since the rescue is partial, it cannot be ruled out that normally the combination of both zfh-1 and eve functions are necessary for EPC development. A putative consensus Zfh-1 homeodomain-binding sequence (P3/RCS1) is present within the eve mesodermal enhancer., and the homeodomain of Zfh-1 can bind to the putative P3/RCSI consensus site in this enhancer. This finding is consistent with the hypothesis that EPC-specific eve expression is under the direct control of Zfh-1 (Su, 1999).

A model is provided of the genetic network regulating the specification and differentiation of the EPC progenitors and their heart and muscle associated progeny (EPC and DA1). Initially, the spatially coincident activity of the transcription factor, Tinman, together with the mesoderm-specific response induced by the patterning signals, Wg and Dpp, are necessary to specify and position the most dorsal portion of the mesoderm, which includes the EPC progenitors and other cardiac precursors. The EPC progenitors then divide and produce two types of progeny cells under the control of the lineage gene numb. The daughter cell that inherits Numb protein will differentiate as the DA1 muscle founder, because the Notch and spdo encoded functions are inhibited, allowing Egf-r signaling (Spitz) to be effective (perhaps in conjunction with Eve). In the daughter cell without Numb, Notch signaling is operational and the transcription factors Zfh-1 together with and/or mediated by Eve can effectively contribute the correct differentiation of the EPC fate. Thus, three levels of information appear to cooperate in the specification of a particular cell fate: prepatterning or positional information, asymmetric lineages and tissue context information (Su, 1999).

Antagonistic function of Lmd and Zfh1 fine tunes cell fate decisions in the Twi and Tin positive mesoderm of Drosophila melanogaster

This study showS that cell fate decisions in the dorsal and lateral mesoderm of Drosophila depend on the antagonistic action of the Gli-like transcription factor Lame duck (Lmd) and the zinc finger homeodomain factor Zfh1. Lmd expression leads to the reduction of Zfh1 positive cell types, thereby restricting the number of Odd-skipped (Odd) positive and Tinman (Tin) positive pericardial cells in the dorsal mesoderm. In more lateral regions, ectopic activation of Zfh1 or loss of Lmd leads to an excess of adult muscle precursor (AMP) like cells. It was also observed that Lmd is co-expressed with Tin in the early dorsal mesoderm and leads to a reduction of Tin expression in cells destined to become dorsal fusion competent myoblasts (FCMs). In the absence of Lmd function, these cells remain Tin positive and develop as Tin positive pericardial cells although they do not express Zfh1. Further, it was shown that Tin repression and pericardial restriction in the dorsal mesoderm facilitated by Lmd is instructed by a late Decapentaplegic (Dpp) signal that is abolished in embryos carrying the disk region mutation dpp^d6 (Sellin, 2009).

Loss of Lame duck (Lmd) leads to an increase of pericardial cells and adult muscle precursor like cells: In embryos lacking Lmd function, staining for zinc finger homeodomain factor 1 (Zfh1) expression reveals a pericardial hyperplasia phenotype and a general excess of Zfh1 positive mesodermal cells. In wild type embryos, three types of pericardial cells (PCs) have been described: Tin positive (TPCs), Odd positive (OPCs) and Eve positive (EPCs) pericardial cells, all of which express Zfh1 and the handC- GFP reporter. Closer inspection of the pericardial cells in lmd mutant embryos revealed that the number of TPCs and OPCs is dramatically increased, while the number of EPCs is normal. All OPCs co-express the handC- GFP reporter and Zfh1 in wild type and lmd mutant embryos. In contrast, a considerable number of ectopic Tin positive cells, though positive for handC- GFP, do not express Zfh1 in lmd mutant embryos. The absence of β3Tubulin expression in these cells is consistent with earlier reports in which a normal set of cardioblasts was described in lmd mutant embryos. To decide whether the Zfh1 negative/Tin positive cells are atypical pericardial cells or dorsal mesodermal cells that fail to differentiate, a triple staining waa conducted for Tin, Zfh1 and Pericardin (Prc), a collagen that is secreted by differentiated pericardial cells. Prc protein was observed surrounding all Tin positive/Zfh1 negative cells, suggesting that they are ectopic pericardial cells. However, due to the fact that Prc is a secreted protein the possibility cannot be ruled out that there might be occasional Tin positive/Zfh1 negative cells in lmd mutant embryos which do not express Prc themselves, but remain in an uncommitted, dorsal mesodermal state (Sellin, 2009).

For further analysis of the ectopic pericardial cells, the number of OPCs in stage 16-17 embryos was counted. An average of 206.3 OPCs was observed in lmd mutant embryos as compared to 97.8 in wild type embryos, thereby representing a ~2-fold increase. It has been reported that the Odd subgroup of pericardial cells (OPCs) originates from two different lineages: a symmetric lineage (two OPCs from one precursor) and an asymmetric lineage (two OPCs from two precursors), adding up to a total of four OPCs per hemisegment. Of note, the siblings of the asymmetrically derived OPCs, the Seven-up (Svp) positive cardioblasts, are normal in lmd mutants, thus suggesting that the asymmetrically derived OPCs do not contribute to the lmd phenotype. Since the two different types of OPCs can not be distinguished directly because the anti-Svp antibody stains the precursor cells and the cardioblast siblings, but not the final PCs at later stages, their abundance was measured in lmd mutants indirectly. The fact was utilized that in inscutable (insc) mutants, asymmetric cell division fails, and all siblings of the asymmetric OPC lineage become Svp positive cardioblasts. The difference in OPC number between insc; lmd and lmd mutant embryos therefore corresponds to the number of asymmetrically derived OPCs in lmd mutant embryos. A loss of ~45 OPCs was observed in insc; lmd double mutant embryos as compared to lmd mutant embryos. This number is reasonably close to the number of ~38 OPCs that are lost in insc mutant embryos when compared to wild type embryos. In addition, the number of Svp positive precursors, which give rise to the asymmetric Odd lineage, is normal in lmd mutant embryos at early stage 13. Altogether, these data strongly support the initial hypothesis that there is the normal amount of asymmetrically derived OPCs in lmd mutant embryos and the phenotype is not caused by a failure of asymmetric cell division (Sellin, 2009).

An excess of Zfh1 positive cells was also observed in the lateral mesoderm of lmd mutant embryos, where it is normally expressed in the adult muscle precursor cells (AMPs). These imaginal myogenic cells retain Twist (Twi) expression, but do not express any other myogenic genes in the embryo. Instead, they are maintained in a less differentiated state during embryogenesis and are dormant until metamorphosis, when they start to differentiate and give rise to the adult musculature of the fly. In the embryo, they are arranged as groups of cells in the thoracic segments, while six solitary cells (one dorsal, two dorsolateral, two lateral and one ventral) are present in the abdominal hemisegments. It was reported earlier that too many Twi positive cells persist in the lateral mesoderm of lmd mutant embryos. Together with the fact that both Zfh1 and Twi are present in AMPs in the wild type, it appeared likely that both factors are also co-localized in embryos mutant for lmd. Indeed, double staining for Zfh1 and Twi showed a complete overlap in the lateral mesoderm and confirmed that both populations of ectopic cells are identical. They also express the gene holes in muscles (him) which is another marker specific for AMPs. For further characterization, the expression patterns were analyzed of several myogenic markers in lmd mutant embryos. No expression was detected of the muscle specific genes myocyte enhancing factor 2 (Mef2), β3 Tubulin or the reporter rP298 (Duf-lacZ) in Twi/Zfh1/Him positive cells (Sellin, 2009).

Twist expression is normally present during early stages of somatic muscle development in myoblasts that are not yet differentiated. Zfh1, which has been implicated in the repression of mef2, might help in keeping AMPs in the undifferentiated state until metamorphosis. The gene him, which is also expressed in AMPs, was recently reported to be involved in maintaining cells in an undifferentiated state by inhibiting the myogenic signal provided by Mef2 function. Consequently, the ectopic Zfh1/Twist/Him positive cells in the lmd mutant embryos are likely to be cells with myogenic potential, as are the endogenous AMPs, and hence can be considered to be ectopic AMP like cells. To assess if enhanced proliferation is also involved in generating an increased amount of cells in lmd mutant embryos, staining was oerfirned for phosphorylated Histone 3 (pH3), which specifically marks dividing cells. Over-proliferation in the dorsal and lateral mesoderm was not observed in lmd mutant embryos when compared to wild type embryos. Although there is a considerable number of the additional, AMP like cells that persist until the end of embryogenesis, their number is reduced between stage 13 and 16/17. Staining with Nile Blue A revealed a general excess of dying cells during these stages in lmd mutant embryos as compared to wild type, suggesting that not all ectopic cells survive until the end of embryogenesis (Sellin, 2009).

The supernumerary PCs and AMPs originate from the population of fusion competent myoblasts: While no general myogenic genes are expressed in the ectopic AMP like cells, it was however possible to show co-localization of Zfh1 and lmd mRNA in the somatic mesoderm of lmd mutant embryos, which is not observed in wild type embryos. In the wild type, lmd is expressed in fusion competent myoblasts (FCMs), which fail to differentiate in the absence of Lmd function. lmd mRNA is transcribed in a normal pattern in lmd mutant embryos. In situ hybridization with a lmd specific riboprobe therefore allowed visualization of the population of cells destined to become FCMs, although they do not express any other FCM specific genes in lmd mutant embryos. Since the ectopic Zfh1 positive cells co-express lmd mRNA, it is concluded that the ectopic AMP like cells in lmd mutant embryos originate from the FCM population and adopt AMP like characteristics instead. They are therefore generated by cell fate conversions, which is consistent with the observation that there are no additional cell divisions in lmd mutant embryos (Sellin, 2009).

In the dorsal mesoderm of lmd mutant embryos, the additional Zfh1 positive cells express Tin or Odd and Prc, indicating differentiation as pericardial cells. Pericardial cells usually develop from the dorsal cardiac mesoderm specified by Tin expression, while the somatic musculature is situated more laterally and is characterized by prolonged Twi expression. To address the question of whether a conversion of FCMs into PCs could also account for the pericardial hyperplasia phenotype in lmd mutant embryos in an analogous fashion to the ectopic AMP like cells, staining was carried out for Tin and lmd mRNA. It was reasoned that the ectopic Tin cells should also express lmd mRNA if they originate from the pool of mis-specified FCMs. Indeed, there is co-expression of lmd mRNA and Tin in ectopic pericardial cells in stage 13 lmd mutant embryos, indicating that cell fate conversions from FCM to ectopic PC fate are responsible for the observed pericardial hyperplasia phenotype (Sellin, 2009).

Of note, there is a distinct overlap of lmd mRNA and Tin expression in the dorsal mesoderm of stage 12 embryos, both in wild type and lmd mutant background. This observation is consistent with the observed cell fate switch from FCM to PC fate and indicates that in wild type embryos, dorsal FCMs are specified in the dorsal, Tin positive mesoderm rather than the Twi positive somatic mesoderm. Indeed, dorsal muscle phenotypes can be observed in embryos mutant for tin, consistent with the conclusion that dorsal muscle cell types (i.e., FCMs) develop from the early dorsal mesoderm specified by Tin expression: If this domain is not specified, it can not generate dorsal FCMs (or other dorsal mesodermal derivatives, like heart or visceral mesoderm) (Sellin, 2009).

Co-expression of Tin and lmd mRNA is no longer detectable after germ band retraction (stage 13) in wild type embryos, but persists in lmd mutant embryos until the lmd mRNA signal fades (at about stage 14-15). Thus, it seems that repression of Tin in the dorsal mesoderm depends on the presence of Lmd protein. To substantiate this observation, Lmd was overexpressed in the whole mesoderm with the twi-Gal4 driver to assess its influence on Tin expression. Indeed, a reduction was observed of Tin expression in stage 12 embryos overexpressing Lmd compared to wild type, further confirming a negative influence of Lmd on Tin expression in the dorsal mesoderm. At later stages, the number of TPCs (and OPCs) remains reduced, while the cardioblasts are not affected. A model is therefore proposed in which the initial dorsal mesoderm specified by Tin expression is subdivided by Lmd into cardiac mesoderm and dorsal musculature by repression of Tin in lateral regions and induction of a myogenic differentiation program instead. During this process, Tin expression is maintained only in the cells that are destined to become pericardial cells (or cardioblasts), while Tin is repressed by Lmd in the dorsally localized FCMs. Loss of Lmd function consistently leads to an increased amount of Tin positive cells in the dorsal mesoderm from stage 13 onwards, which then can differentiate as ectopic pericardial cells as indicated by the expression of Prc. Taken altogether, these data suggest that, in the absence of Lmd function, the pool of unspecified FCMs can develop as ectopic PCs in the Tin-positive dorsal mesoderm and as AMP-like cells in the lateral and ventral mesoderm. However, increased cell death, and the possibility that a small number of ectopic Tin positive cells might exist without Prc/Zfh1 expression as mentioned earlier, suggest the possibility that not all cells of the FCM population follow alternative cell fates. Instead, some cells might remain in an uncommitted mesodermal state in lmd mutant embryos (Sellin, 2009).

Normally, instructive Dpp signals from the ectoderm are responsible for the specification of cardiac cell types by maintaining Tin expression solely in the dorsal mesoderm, while Twist activity in the lateral and ventral mesoderm leads to the development of the somatic musculature. To test if reduced Dpp signaling has a similar effect on PC number as overexpression of Lmd, by reducing the size of the Tin domain, embryos carrying the mutation mad^1-2 were examined. mad^1-2 is a weak hypomorphic allele of the Dpp effector Mad and causes larval lethality, thereby allowing observation of late stages of embryogenesis. Indeed, a decreased number of OPCs and TPCs was observed in mad^1-2 mutant embryos, without any effect on cardioblast number, as is the case when overexpressing Lmd. Of note, the number of OPCs is decreased to a similar extent in mad^1-2; lmd double, as compared to lmd single mutant embryos. Therefore, it is concluded that in the presence of the hypomorphic mad^1-2 mutation, the dorsal mesoderm that is specified by Dpp-dependent Tin expression is reduced, resulting in a reduction of PCs in a Lmd independent manner. However, Lmd further restricts the number of PCs in the mad^1-2 mutant background, as revealed by an increased number of PCs and the presence of TPCs without Zfh1 expression in mad^1-2; lmd double mutants when compared to mad^1-2 single mutants (Sellin, 2009).

Pericardial cells share their developmental origin with the myogenic cardioblasts in a similar fashion as AMPs with founder cells in the somatic musculature. During lateral inhibition, Notch activation promotes myogenic FCM fate as opposed to the progenitors of founder cells in the lateral mesoderm or cardiogenic progenitors in the dorsal mesoderm. Subsequently, during the process of asymmetric cell division, Notch activation renders the daughter cell always non-myogenic (PC or AMP fate). Although the AMPs have the potential to develop into muscle cells during metamorphosis, they are considered non-myogenic in this context because they do not yet express any myogenic genes, such as mef2, lmd or muscle structural genes in the embryo. In the case of pericardial cells, there is surprisingly little data available about their physiological role. While it is known that the OPCs contribute to the population of nephrocytes in postembryonic stages, TPCs and EPCs are not correlated with any function at all, and their developmental fate after embryogenesis is still unknown. A recent study described the development of adult muscular structures, the so called wing hearts, from a specialized subset of EPCs. This is the first hint that some pericardial cells might be considered as imaginal myogenic cells in an analogous fashion to AMPs, and it highlights the necessity to further characterize pericardial cells (Sellin, 2009).

It is currently known that PCs and AMPs have in common a dependency on active Notch signaling although they stem from different cell lineages and mesodermal primordia (Tin vs. Twi domain). FCMs, which adopt AMP or PC like characteristics in lmd mutant embryos, also need active Notch signaling. In fact, Lmd is a downstream target of N signaling and induces the FCM differentiation program. The observed lmd phenotype could be explained if, in the absence of Lmd, Notch activity always promoted AMP or PC (non-myogenic) fate, but not FCM fate, independently of the original pathway that is involved (lateral inhibition or asymmetric cell division). To assess this hypothesis, double mutants for lmd and genes involved in the Notch pathway were established. For this analysis kuzbanian and mastermind alleles were chosen because loss of either gene causes lethality only late in embryogenesis due to a maternal component, thereby allowing the analysis of later events in heart and muscle development. Both genes have also been well studied with respect to their molecular function and developmental implications. Kuzbanian (Kuz) is an ADAM metalloprotease that is known to process the Notch receptor following ligand binding. Zygotic loss of function mutations lead to defects in both lateral inhibition and asymmetric cell division in heart and muscle development, although the phenotype is far weaker than in embryos carrying N loss of function alleles. mastermind (mam) is involved in transducing the Notch signal and displays a stronger heart phenotype than kuz and a mild Notch-like muscle phenotype. Staining was perfomed for expression of Krüppel (Kr) and him mRNA, which are specific for a subset of muscle founders and AMPs/ PCs, respectively, and an increase of Notch negative cell types, corresponding to founders, was observed in the somatic mesoderm of kuz mutant embryos. This is accompanied by a reduction of AMPs, confirming the expected function of Kuz in facilitating N function in muscle cell differentiation. Furthermore, the number of FCMs as marked by Lmd expression is strongly reduced in kuz mutants, although the effect is not as complete as in N loss of function alleles (Sellin, 2009).

In kuz; lmd double mutant embryos, the increase of AMPs is milder than in lmd mutant embryos, which is consistent with a failure in lateral inhibition and a concomitant reduction of FCMs that are available for conversion to AMPs. The number of Kr-positive founder cells is increased to comparable levels in kuz and kuz; lmd mutant embryos, suggesting that Notch inactive cell fates (muscle founders and cardioblasts) are not influenced by the absence of Lmd, and that Notch acts as a permissive signal to allow the cell fate switch in lmd mutant embryos. mam; lmd double mutant embryos display a similar phenotype. Altogether, these findings suggest that in the double mutants, a general reduction of cell types with Notch activity (i.e. FCMs) occurs, followed by the conversion of the remaining potential FCMs to AMP or PC fate under the influence of N signaling in the absence of Lmd. Lame duck is present in stages 12-14, which is later than the period during which Notch activity is involved in facilitating cell fate decisions within the musculature. Hence, it appears that Notch can promote AMP or PC fate at a relatively late time point in the absence of Lmd (Sellin, 2009).

It was of interest to know if the endogenous set of AMPs, which develop through asymmetric cell divisions of muscle progenitors, is specified correctly in lmd mutant embryos. For example, the lateral AMPs are the siblings of the segment border muscle founder (SBM), and share with the latter the expression of the identity factor Ladybird early (Lbe). To discern ectopic cells and endogenous AMPs in lmd mutant embryos, co-staining was performed for Lbe and Twi expression. Indeed, the normal number of lateral AMPs, as marked by Lbe expression, is present in lmd mutant embryos, while far too many Twi-positive cells was observed in general. The latter are the ectopic AMP like cells that are presumed to be recruited from the FCM population. This observation further confirms that individual mesodermal lineages, such as the asymmetrically derived OPCs or individual AMPs, are not influenced by the loss of Lmd function (Sellin, 2009).

The proposed model of cell fate switches from myogenic FCM fate to non-myogenic AMP like or PC fate, but not myogenic fates (cardioblasts or founder cells), is consistent with the observation that Notch signaling is often employed to delay or inhibit the differentiation of stem cells or progenitor cells, especially in myogenesis. In vertebrates, Notch signaling prevents satellite cells (muscle stem cells) from entering a myogenic differentiation program in cell culture as well as in vivo, and impaired upregulation of its ligand Delta-like 1 in satellite cells has been correlated with a decreased capacity of aging muscle tissue to regenerate. While the data are consistent with the general function of Notch in preventing cells to enter the myogenic differentiation program by promoting the AMP or PC fate, they also highlight the special and unusual properties of Lmd - as a target of Notch signaling - in Drosophila muscle development. Although it is activated by Notch, it has the ability to induce myogenic differentiation. The data strongly suggest that the AMP or PC fate is the default consequence of Notch signaling in Drosophila myogenesis and that Lmd function overrules this signal to induce the FCM differentiation program in lateral or dorsal competence domains. It was shown that N has a biphasic function in heart differentiation analogous to the situation in the somatic mesoderm. At an early phase, N activity restricts the number of the sum of CBs and PCs, reflecting a function in the definition of early cardiac progenitors, likely by lateral inhibition. Subsequently, N activity is needed to promote pericardial cell fates in asymmetric cell division of the early progenitors. Although the last division step is in many cases a symmetric division seem to indicate that the majority of cardiac cell types is generated by asymmetric cell divisions segregating cardiac and pericardial fates. This might occur in some cases at one of the earlier division steps of the progenitor(s). Since these data indicate the generation of FCMs from the dorsal mesoderm, as reflected by co-expression of Tin and Lmd in stage 12 embyos, it might be suggested that dorsal FCMs originate from dorsal competence domains which also give rise to the above mentioned cardiac progenitors. These progenitors divide asymmetrically to generate CBs and PCs analogous to FC/AMP sibling pairs from more lateral competence fields, while it ia proposes that all or some of the remaining cells of the competence domains begin to express Lmd and generate FCMs under instructive influence of N signaling. In the absence of Lmd function (either in wild type in the N active daughter cells of the progenitors, or in lmd mutant embryos in all N active cells of the competence domains), the N signal promotes non-myogenic cell fates according to the mesodermal context (i.e., dorsal vs. lateral mesoderm). This would then result in the differentiation of the non-segregating population (normally developing as FCMs) as PCs in the Tin domain and AMPs in the somatic mesoderm (Sellin, 2009).

Lame duck and Zfh1 act antagonistically in mesodermal cell fate decisions: While loss of Lmd function results in an increased number of Zfh1-positive cell types, overexpression of Lmd leads to the opposite phenotype. The pan-mesodermally active twi-Gal4 driver line was used to induce Lmd expression in the whole mesoderm, and a reduction of OPCs, TPCs and AMPs was observed. To assess whether pericardial cell reduction might be a secondary effect of the early Tin repression caused by ectopic Lmd activity, the later and cardiac specific handCA-Gal4 driver, which is active in the heart from stage 12 onwards, was used. At this time point, the OPC precursors are already specified and are no longer expressing Tin. Since hand>Lmd overexpression severely reduces the number of all pericardial cells, it is concluded that their reduction is not only a secondary effect of the narrower Tin domain in embryos overexpressing Lmd. To further confirm this conclusion, the phenotype of zfh1 mutant embryos was compared with that of embryos overexpressing Lmd. The number of OPCs and TPCs is also reduced in zfh1 mutant embryos quite similarly to embryos overexpressing Lmd, although the early Tin expression pattern is normal in the absence of Zfh1 function. It is therefore unlikely that Lmd acts negatively on Zfh1 expression only by reducing Tin expression, but rather also independently of Tin function (Sellin, 2009).

There are however important differences in the phenotypes of twi > Lmd and zfh1 mutant embryos. Zfh1 appears to be involved in maintaining, but not in specification of OPCs, because it has been observed that loss of Zfh1 does not affect the number of OPC precursors at stage 13, but rather leads to a decrease of OPCs at later stages. This is in contrast to a reduced number of OPC precursors in stage 13 embryos overexpressing Lmd. Therefore, Zfh1 repression alone can not account for the loss of PCs in embryos ectopically expressing Lmd. Instead, it might be that the reduction of the dorsal Tin domain by ectopic Lmd expression results in the specification of fewer OPC precursor cells, followed by further reduction of the remaining OPCs by the negative effect of ectopic Lmd on Zfh1 expression. Consistently, a much stronger reduction of OPCs was observed after ectopic expression of Lmd as compared to the loss of OPCs in zfh1 mutant embryos. The observation that loss of Lmd function leads to the appearance of TPCs that do not express Zfh1, but Prc as a marker of pericardial differentiation, is another hint that both effects occur independently of each other and that pericardial differentiation can be accomplished in the absence of Zfh1 in lmd mutant embryos (Sellin, 2009).

Taken altogether, it does not seem likely that Tin and Zfh1 act in an epistatic hierarchy in dorsal mesodermal cell fate decisions. Instead, the data support the conclusion that Lmd regulates OPC and TPC number by two independent mechanisms: (1) Initially, Lmd restricts the cardiac field in general through repression of Tin, which leads to the reduction of early OPC precursors and the elimination of Tin expression in cells that do not express Zfh1 (which can differentiate as TPCs, as indicated by Prc expression, in the absence of Lmd function). (2) Later, it represses Zfh1, thereby reducing further the number of OPCs and TPCs. This is consistent with previous findings which described Zfh1-dependent and Zfh1-independent mechanisms for the regulation of OPC and TPC number (Sellin, 2009).

Of note, it was previously shown that Zfh1 overexpression leads to an increase in pericardial cell number (both OPCs and TPCs) and a concomitant loss of dorsal somatic muscle cells, indicating that overexpression of Zfh1 phenocopies the pericardial hyperplasia in lmd mutant embryos. It was shown further that overexpression of Zfh1 with the twist-Gal4 or 24B-Gal4 driver leads to an increased number of AMP like cells in the dorsal mesoderm although the effect is rather weak when compared to lmd mutant embryos. Zfh1 overexpression does not however alter the pattern of Lmd expression, indicating that Zfh1 does not antagonize Lmd function at the transcriptional level. To verify whether Zfh1 has an influence on Lmd at the posttranscriptional level, the intracellular distribution of Lmd was analyzed in embryos overexpressing Zfh1, because Lmd function has been shown to be modulated by its subcellular localization in wild type embryos. In embryos overexpressing Zfh1, the subcellular localization of Lmd does not appear to be altered, suggesting that Zfh1 does not influence the subcellular distribution of the Lmd protein (Sellin, 2009).

Taken together, these data indicate that Lmd and Zfh1 have generally opposite effects on dorsal mesoderm differentiation: Lmd loss-of-function or Zfh1 gain-of-function leads to increased AMPs or PCs, whereas Lmd gain-of-function and Zfh1 loss-of-function reduce these cell types. Consequently, Lmd and Zfh1 can be considered to be functional antagonists, although their repression is not mutual. One possible explanation for the antagonistic effect of Zfh1 overexpression might be due to its direct negative influence on mef2 expression, thereby counteracting the mef2 activating function of Lmd. The vertebrate functional orthologue of Zfh1, ZEB2 (or Sip1), also inhibits myotube development in culture and represses a number of myogenic genes, and is able to rescue Zfh1 function in Drosophila (Sellin, 2009).

Lmd is instructed to restrict Tin expression by a late, pro-myogenic Dpp signal: While in wild type embryos Tin expression is repressed in cells destined to become dorsal FCMs between stages 12 and 13, there is a prolonged co-localization of Tin and lmd mRNA in cells of the dorsal mesoderm in lmd mutant embryos. As a consequence, dorsal FCMs adopt pericardial cell fates in the absence of Lmd function. Of note, this effect can also be observed in embryos carrying the dpp^d6 disk region mutation. These embryos lack a late Dpp signal (beginning at about stage 12) that is involved in pericardial restriction. Early Dpp signaling does not seem to be affected since the dorsal mesoderm (characterized by Dpp-dependent Tin expression) is normal in dpp^d6 mutant embryos. Quite contrary to embryos with otherwise decreased Dpp signaling and a reduced pericardial field, such as mad^1-2 embryos, the dpp^d6 mutant embryos display a pericardial hyperplasia phenotype that resembles in many aspects the phenotype observed in lmd mutant embryos. Too many OPCs, TPCs and atypical TPCs without Zfh1 expression are also detected, although the dpp^d6 mutant phenotype is milder than the lmd mutant phenotype. This resemblance in phenotypes suggested an epistatic relationship of Lmd and the late Dpp signal. In addition, the accumulation of phosphorylated Mad (pMad) has been traced in PCs and cells within the dorsal musculature that are not positive for founder specific Kr or Eve expression, and hence are likely to be FCMs. Altogether, these findings lead to the hypothesis that Lmd might be a target of the late Dpp signal in FCMs. However, Lmd is expressed in a normal pattern (both at the mRNA and protein levels) in dpp^d6 mutant embryos, indicating that Lmd expression is independent of Dpp signaling. Nevertheless, co-staining with anti-Tin antibody revealed a prolonged co-localization of Tin and lmd mRNA in dpp^d6 mutant embryos until stage 14/15, as observed in lmd mutant embryos, suggesting a requirement for late Dpp signaling in the process of pericardial restriction by Lmd. To assess if the restrictive influence of late Dpp signaling on Tin expression is indeed relayed by Lmd in the dorsal mesoderm, or if both negative effects are independent of each other, the late Dpp signal was enhanced in the lmd mutant background. For this purpose, the leading edge driver LE-Gal4 was used to overexpress Dpp, which was shown to reduce the number of OPCs and TPCs in the wild type background. It was reasoned that this effect would be lost in lmd mutant embryos if Lmd is responsible for the restricting effect on PC number. The number of OPCs was counted in LE > Dpp; lmd embryos in comparison to lmd mutant embryos. While overexpression of Dpp with the LE-Gal4 driver in the wild type background led to a reduction of OPCs by ~1.2-fold, no reduction of OPCs was observed in the lmd mutant background, indicating that Lmd is indeed necessary to interpret the late Dpp signal as pro-myogenic. Altogether, these data suggest that the pro-myogenic effect of the late Dpp signal is Lmd dependent, although not by inducing Lmd expression. Instead, the presence of Dpp activity seems to be a prerequisite for the negative influence of Lmd on Tin expression and might act as an instructive signal to modify Lmd activity to allow repression of Tin. If the late Dpp signal is lost -- as is the case in embryos carrying the hypomorphic allele dpp^d6 - repression of Tin fails even in the presence of Lmd protein, indicating that repressive activity of Lmd is dependent on Dpp signaling (Sellin, 2009).

A model is proposed in which the subdivision of the early Tin positive primordium into pericardial and dorsal muscle tissues is mediated via the antagonistic action of Lmd and Zfh1 under the instructive influence of late Dpp signals. While the early function of Dpp restricts Tin expression to the dorsal mesoderm, subsequent Dpp signaling provides pro-myogenic input to modulate the pericardial field in favor of the dorsal musculature. The present data show that the function of this late Dpp signal requires Lmd activity, strongly suggesting that Lmd is a target of Dpp for establishing the boundary between the dorsal musculature and pericardial field. Repression of Tin also appears to be dependent on Dpp signaling. The previous observation that pMad accumulation occurs in PCs and dorsal muscle cells, which are likely to be FCMs, is consistent with the finding that Lmd is needed to relay the pro-myogenic function of late Dpp signaling. These cells originate from the Tin-expressing dorsal mesoderm, and co-expression of Tin and lmd mRNA in wild type embryos at stage 12 can be observed. In the presence of Lmd protein, this co-expression is not maintained after stage 12 due to a repressive function of Lmd on Tin. Of note, it was previously shown that Lmd function depends on posttranscriptional mechanisms that modulate its specific subcellular localization and activity, and it might be speculated that Dpp signaling is involved in changing Lmd function into a repressive form. However, there is no evidence that the negative influence of Lmd on Tin expression is of a direct nature, or if there are other factors that are involved in the process. In this context, the following explanation for the antagonistic effect of Zfh1 overexpression without repression of Lmd could also be considered. Since the vertebrate homologue ZEB2 was shown to inhibit activation of target genes by Smads, an excess of Zfh1 might antagonize the late Dpp signal by repressing pMad-dependent interaction partners of Lmd, thereby preventing the repression of Tin (and/or other targets) in the dorsal mesoderm. Lmd expression and function would not be affected elsewhere which would be consistent with the observation that Zfh1 is not a general repressor of Lmd (Sellin, 2009).

Decoding the regulatory logic of the Drosophila male stem cell system

The niche critically controls stem cell behavior, but its regulatory input at the whole-genome level is poorly understood. This study elucidated transcriptional programs of the somatic and germline lineages in the Drosophila testis and genome-wide binding profiles of Zfh-1 and Abd-A expressed in somatic support cells and crucial for fate acquisition of both cell lineages. Key roles were identified of nucleoporins and V-ATPase proton pumps, and their importance was demonstrated in controlling germline development from the support side. To make the dataset publicly available, an interactive analysis tool was generated, that uncovered conserved core genes of adult stem cells across species boundaries. The functional relevance of these genes was tested in the Drosophila testis and intestine, and a high frequency of stem cell defects was found. In summary, this dataset and interactive platform represent versatile tools for identifying gene networks active in diverse stem cell types (Tamirisa, 2018).

Using cell-type-specific transcriptome profiling and in vivo TF binding site mapping together with an interactive data analysis tool, this study comprehensively identified genes involved in controlling proliferation and differentiation within a stem cell support system. Importantly, many candidates that were functionally tested not only were required within the soma, but also had non cell-autonomous functions in the adjacent (germline) stem cell lineage (Tamirisa, 2018).

An interconnected network of TFs was identified that plays an important role in the maintenance and differentiation of both germline and somatic cell populations, signal processing V-ATPase proton pumps, and nuclear-transport-engaged Nups as regulators in the Drosophila male stem cell system. V-ATPases have been implicated in the regulation of various cellular processes in not only invertebrates but also vertebrates. For example, the V-ATPase subunit V1e1 was previously shown to be essential for the maintenance of NBs in the developing mouse cortex, as loss of this subunit caused a reduction of endogenous Notch signaling and a depletion of NBs by promoting their differentiation into neurons. Furthermore, two independent studies revealed that V-ATPase subunits and their isoforms are required for proper spermatogenesis in mice, in particular for acrosome acidification and sperm maturation. Thus, it is tempting to speculate that these proton pumps also have important functions in the stem cell pool of the mammalian testis and very likely many other stem cell systems, and some evidence is provided for their crucial role also in ISCs (Tamirisa, 2018).

This work also uncovered nuclear transport associated proteins, the Nups, as important control hubs in the somatic lineage of the Drosophila testis. This is of particular interest, since cell-type-specific functions of Nups have been identified only recently and may represent a critical feature of different stem cell systems. Examples include Nup153, one of the Nups expressed in all four stem cell systems, which interacts with Sox2 neural progenitors and controls their maintenance as well as neuronal differentiation; Nup358, which plays a role at kinetochores, and Nup98, which regulates the anaphase promoting complex (APC) and mitotic microtubule dynamics to promote spindle assembly. Interestingly, it has been shown just recently that Nups play a critical role in regulating cell fate during early Drosophila embryogenesis, thereby contributing to the commitment of pluripotent somatic nuclei into distinct lineages. Current results suggest that they may play a similar role in controlling the transition of continuously active adult stem cells toward differentiation. The next challenge will be to unravel how variations in the composition of an essential and basic protein complex like the NPC causes differential responses of cells, in particular in stem cells and their progenies (Tamirisa, 2018).

The datasets in conjunction with the versatile and easy-to-use analysis tool allowed identification of a substantial number of stem cell regulators for detailed mechanistic characterization. Importantly, this analyses have shed first light on processes and genes shared between diverse invertebrate and vertebrate stem cell systems and uncovered functionally relevant differences. Owing to its flexibility and the option to include datasets from any species, the online tool represents a valuable resource for the entire stem cell community. It not only provides an open platform for data analysis but also leverages the power of comparative analysis to enable researchers mining genomic datasets from diverse origins in a meaningful and intuitive fashion (Tamirisa, 2018).

Notch mediates inter-tissue communication to promote tumorigenesis

Disease progression in many tumor types involves the interaction of genetically abnormal cancer cells with normal stromal cells. Neoplastic transformation in a Drosophila genetic model of Epidermal growth factor receptor (EGFR)-driven tumorigenesis similarly relies on the interaction between epithelial and mesenchymal cells, providing a simple system to investigate mechanisms used for the cross-talk. Using the Drosophila model, this study shows that the transformed epithelium hijacks the mesenchymal cells through Notch signaling, which prevents their differentiation and promotes proliferation. A key downstream target in the mesenchyme is Zfh1/ZEB. When Notch or zfh1 are depleted in the mesenchymal cells, tumor growth is compromised. The ligand Delta is highly upregulated in the epithelial cells where it is found on long cellular processes. By using a live transcription assay in cultured cells and by depleting actin-rich processes in the tumor epithelium, this study provides evidence that signaling can be mediated by cytonemes from Delta-expressing cells. It is thus proposed that high Notch activity in the unmodified mesenchymal cells is driven by ligands produced by the cancerous epithelial. This long-range Notch signaling integrates the two tissues to promote tumorigenesis, by co-opting a normal regulatory mechanism that prevents the mesenchymal cells from differentiating (Boukhatmi, 2020).

Normal tissue mesenchymal cells are thought to have important roles in promoting the growth and metastasis of many tumors. To do so, they must be educated by the aberrant cancerous cells to acquire the properties needed to sustain tumorigenesis. Using a Drosophila model of EGFR/Ras-driven tumorigenesis, this study demonstrates that Notch activity in the unmodified mesenchymal cells is essential for tumor growth. Downregulating Notch specifically in mesenchymal cells reduced their proliferation rates, promoted their differentiation, and significantly compromised the size of tumors that developed. Strikingly, the activation of Notch in these supporting cells appears to rely on direct communication from the cancerous epithelial cells, illustrating that this pathway can operate in long-range signaling between tissue layers (Boukhatmi, 2020).

The conclusion that Notch receptors in the mesenchymal cells are activated from ligands presented by nearby epithelial cells is unexpected because most examples of Notch signaling occur between cells within an epithelial cell layer. The fact that the ligands are transmembrane proteins means that direct cell-cell contacts are required to elicit signaling and that signaling usually occurs between neighboring cells. More recently, examples have emerged where signaling occurs across longer distances that appear to involve contacts mediated by cell protusions, such as filopodia or cytonemes. Evidence indicates that a similar mechanism operates in the tumors. Delta is produced in the epithelial cells and can be detected in fine processes that extend through the nearby mesenchymal cells, which is consistent with a recent report describing cytonemes in these EGFR-psqRNAi tumors. In a heterologous system, it was found there was robust activation of a Notch target gene rapidly after ligand-expressing cells made contact through cell processes. Likewise, ectopic patches of Delta in the disc epithelium led to the expression of the Notch-regulated m6-GFP in the underlying mesenchyme. Thus, it is proposed that the widespread upregulation of Delta in the epithelial compartment of the tumorous wing discs, in turn, activates the Notch pathway in the neighboring mesenchymal cells by long cellular processes. As a consequence, the mesenchymal cells become coordinated with the cancer epithelial cells and are maintained in an undifferentiated state (Boukhatmi, 2020).

Although the data demonstrate that Delta-Notch-mediated inter-tissue signaling is important for sustaining tumor growth, it is evident that other signals are also required. First, it was previously shown that Dpp from the cancerous epithelium is essential for these tumors to grow. Because the Dpp pathway was still activated in the mesenchyme when Notch was depleted, it is proposed that Dpp and Notch operate in parallel. This may explain why apicobasal polarity was not fully restored when Notch activity was impaired and highlights the likelihood that several different pathways are coopted to drive tumorigenesis. Second, the fact that tumorigenesis is rescued by perturbing Notch or Dpp signaling in the mesenchyme argues that there must be a reciprocal signal to the epithelium. Notably, the relative growth of the two populations appears highly co-ordinated in the tumors, unlike the wild type where the epithelial growth predominates. A plausible model is that combined inputs from Notch and Dpp are required to produce reciprocal signal(s), and it will be interesting to discover whether the reciprocal signaling also operates through cytonemes, given that the mesenchymal cells emit processes (Boukhatmi, 2020).

One of the key effectors of Notch activity in the tumor mesenchyme is Zfh1/ZEB, which is important for maintaining the muscle progenitors in normal conditions. In a similar manner, its expression is kept high in the tumor mesenchyme, due to Notch activity, where it helps prevent their differentiation. Downregulating zfh1 in mesenchymal cells induces their premature differentiation and prevents tumor growth. The role of Zfh1/ ZEB in promoting progenitors and stem cell proliferation appears to be widespread. Furthermore, ZEB1 is upregulated in many cancers, where it can cause the expansion of cancer stem cells and frequently drives epithelial-to-mesechymal transition to promote metastasis. Whether its activation in these conditions also involves Notch activation and inter-tissue signaling remains to be determined (Boukhatmi, 2020).

GENE STRUCTURE

Bases in 5' UTR - 357

Bases in 3' UTR - 1753

PROTEIN STRUCTURE

Amino Acids - 1060

Structural Domains

Drosophila ZFH-1 has one homeodomain and nine C2-H2 zinc fingers in contrast to the Drosophila protein ZFH-2 with three homeodomains and sixteen C2-H2 zinc fingers. The two proteins can be considered divergent homologs: their homeodomains are both more similar to ATBF1 homeodomains (ATBF1 is a human zinc finger homeodomain protein) than than they are to each other (Hashimoto, 1992). Comparison of each individual homeodomain sequence of Drosophila ZFH-1 and ZFH-2 to other homeodomain sequences indicates that the closest match is to mec-3, a LIM homeodomain expressed in mechanosensory neurons in C. elegans (See Drosophila Islet). Drosophila ZFH-1 contains two isolated fingers, a cluster of four fingers about a third of the way through the protein and a C-terminal cluster of three fingers. Alignment of the ZFH-1 zinc-finger sequences reveals that in their central portions, fingers 3-5 resemble fingers 7-9. This similarity, together with the tandem arrangement of each set of three fingers, raises the possibility that the different zinc-finger regions of ZFH-1 protein were generated by a gene duplication event. In comparison to Drosophila ZFH-1, the fingers of Drosophila ZFH-2 are less clustered (Fortini, 1991). All zinc fingers in ATBF1 and Drosophila Zfh proteins belong to the C2-H2 type except for two each in ATBF1 and Drosophila ZFH-1, which are of the C2HC class, and one in ZFH-2, which has a SCH2 arrangement (Hashimoto, 1992).

The third homeodomain of Drosophila ZFH-2 most closely resembles the one homeodomain of Drosophila ZFH-1. The first homeodomain of ZFH-1 may be a nonfunctional "pseudohomeodomain," since two unorthodox amino acid residues in the homeodomain may prevent binding to DNA. Both proteins possess regions rich in certain amino acid residues, notable alanine, glutamic acid, serine, proline and glutamine. A particularly long run of glutamines in ZFH-1 is encoded by an opa repeat, a motif of uncertain function associated with Drosophila homeobox genes (Fortini, 1991).

The homeodomains of ATBF1 show 32% or lower sequence identity with the Antennapedia-class homeobox sequences, indicating the divergent nature of these homeodomains. In contrast, the third homeodomain of ATBF1 is 51% identical with the Drosophila ZFH-1 homeodomain, indicating a closer affinity of the homeodomains of the zinc-finger homeodomain to each other than to the Antennapedia-class homeodomain. Higher levels of sequence conservation are observed between ATBF1 and Drosophila ZFH-2 homoeodomains. The first three homeodomains of ATBF1 share 77, 69 and 61% identity with the corresponding homeodomains of AFH-2. The more C-terminal fourth homeodomain of ATBF1 shows a 46% identity with ZFH-2 third homeodomain. ZFH-1 and ZFH-2 homeodomains homologies are less than 39%, indicating that they are more similar to human ATBF1 homeodomains than to one another (Hashimoto, 1992).

The entire coding region of ZFH-1 is present in some of the larger cDNA clones analyzed. RNA blot analysis of embryos detects a single AFH-1 transcript of 7.5 kb and three ZFH-2 transcripts of 10.5, 11 and 13 kb. Nevertheless, these data cannot rule out the possibliity that the open reading frames encode large precursor proteins that are processed into polypeptides bearing single DNA-binding domains. Different anti-ZFH-1 sera all recognize a common polypeptide species that migrates on denaturing gels with an apparent molecular weight of 145 kDa (which is only slightly larger than the 117 kDa expected for the zfh-1 long open reading frame sequence). These results strongly suggest that the mature zfh-1 gene product contains both the homeodomain and the zinc fingers predicted by its DNA sequence. As sera representing five nonoverlapping regions of the ZFH-2 protein display identical staining patterns in whole-mount embryos, it is thought that the zfh-2 long open reading frame is translated into a single large protein (Fortini, 1991).

date revised: 14 April 98  

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