Gene name - Zn finger homeodomain 1
Cytological map position - 100A3--100A4
Function - transcription factor
Keyword(s) - neural, mesoderm
Symbol - zfh1
FlyBase ID: FBgn0004606
Genetic map position - 3-
Classification - homeodomain, zinc finger domains
Cellular location - nuclear
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 (eveID), which produces a non-functional but nevertheless antigenic protein at the non-permissive temperature, was used. When eveID 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 eveID 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).
Bases in 5' UTR - 357
Bases in 3' UTR - 1753
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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