Gene name - elbow B
Cytological map position - 35A3
Function - putative transcription factor
Keywords - tracheal cell identity
Symbol - elB
FlyBase ID: FBgn0004858
Genetic map position - 2-50.0
Classification - Sp1-type zinc finger
Cellular location - nuclear
The elbow B (elB) gene encodes a conserved nuclear protein with a single zinc finger. Expression of ElB is restricted to a specific subset of tracheal cells, namely the dorsal branch and the lateral trunks. Stalled or aberrant migration of these branches is observed in elB mutant embryos. Conversely, ElB misexpression in the trachea gives rise to absence of the visceral branch and an increase in the number of cells forming the dorsal branch. These results imply that the restricted expression of ElB contributes to the specification of distinct branch fates, as reflected in their stereotypic pattern of migration. Since elB loss-of-function tracheal phenotypes are reminiscent of defects in Dpp signaling, the relationship between ElB and the Dpp pathway was examined. By using pMad antibodies that detect the activation pattern of the Dpp pathway, it has been shown that Dpp signaling in the trachea is not impaired in elB mutants. In addition, expression of the Dpp target gene kni is unaltered. The opposite is true as well, because expression of elB is independent of Dpp signaling. ElB thus defines a parallel input, which determines the identity of the lateral trunk and dorsal branch cells. No ocelli (Noc) is the Drosophila protein most similar to ElB. Mutations in noc give rise to a similar tracheal phenotype. Noc is capable of associating with ElB, suggesting that they can function as a heterodimer. ElB also associates with the Groucho protein, indicating that the complex has the capacity to repress transcription of target genes. Indeed, in elB or noc mutants, expanded expression of tracheal branch-specific genes is observed (Dorfman, 2002).
The Drosophila tracheal system is a stereotypical network of interconnected tubes that supplies air to all cells of the organism. Initially, ten tracheal placodes are defined on both sides of the embryo, each consisting of 20 cells. The placodes undergo two rounds of division, giving rise to the final number of tracheal cells. All subsequent events of tracheal morphogenesis and branch migration occur in the absence of any further cell division. The final structure of the tracheal tree is elaborate. Each tracheal pit gives rise to five different branches: dorsal branch (DB), dorsal trunk (DT), visceral branch (VB), lateral trunk anterior (LTa) and lateral posterior/ganglionic branch (LTp/GB). The number of cells allocated to each branch is fixed and the final structure of each branch is stereotyped, reflecting established migration routes. Within each branch, different cell types are formed from an originally equipotent population of tracheal cells. The cells at the termini of the branches differentiate as terminal cells that send long hollow extensions to hypoxic tissues. Another group of specialized cells, termed fusion cells, establishes connections between branches from adjacent segments (Dorfman, 2002 and references therein).
This elaborate tracheal structure is set up by the concerted activity of multiple signaling pathways. The initial assignment of tracheal fates within the population of ectodermal cells is driven by the localized expression of the Trachealess and Drifter transcription factors. Persistent expression of these genes in the trachea provides a 'cell context' for other signals that impinge on the trachea. Prior to the onset of tracheal migration, the precise number of cells must be allocated to each future branch. Several signaling pathways contribute to this decision, and in many cases parallel inputs from different pathways are responsible for the assignment of a particular branch fate. The process of migration is guided by the FGF pathway. All tracheal cells express the FGF receptor, Breathless (Btl). The ligand, Branchless (Bnl), is expressed locally in adjacent ectodermal or mesodermal cells. This restricted ligand presentation is responsible for guided migration. In addition, as the branches elongate, the levels of Btl activation determine the fate of the cells as terminal or fusion cells. Additional accessory guidance systems are present, such as the presence of a mesodermal cell expressing Hunchback (Hb) that assists the migration of the dorsal trunk cells (Dorfman, 2002 and references therein).
Morphogenesis of the tracheal system is determined by highly coordinated signaling events, which are restricted in both space and time. This prompted a search for new genes regulating tracheal development using the EP misexpression screen. The midline- and tracheal-specific btl-Gal4 driver was used, and the collection of EP lines was screened for those that give rise to lethality because of aberrant development of the tracheal system, or other tissues expressing btl-Gal4. Known genes that regulate tracheal patterning, such as dpp, bnl, rhomboid and escargot were scored, validating the specificity of the approach. In addition, this screen identified new genes that were not previously known to be involved in patterning the trachea. ElB defines a pathway acting parallel to Dpp that determines the identity of the lateral trunks and dorsal tracheal branches (Dorfman, 2002).
ElB is a member of a new family of proteins containing a single zinc finger and additional conserved motifs. ElB is a nuclear protein, but it is not yet known whether it binds DNA, or if it functions as a monomer or multimer. The similarity between elB and noc mutant phenotypes, the genetic interactions between them (Davis, 1997), and the ability of ElB and Noc proteins to associate with each other, suggest that ElB/Noc heterodimers are the functional complex (Dorfman, 2002).
ElB overexpression represses expression of genes such as the visceral branch marker and spalt (sal). Conversely, absence of ElB results in expanded expression of Sal to the dorsal branch, and noc mutants display failure to repress Serum response factor (SRF) expression in the fusion cells of the dorsal branch, suggesting that ElB functions as a repressor of gene expression. One piece of evidence strongly indicates that the ElB/Noc complex indeed functions directly to repress the expression of target genes. Both Drosophila proteins, as well as the human homologs, contain the FKPY motif, which is known to be sufficient for interactions with Groucho. Indeed, GST pull-down experiments have demonstrated that ElB can associate with Groucho. It is interesting to note that another Sp1 homolog, Huckebein (Hkb), recruits Groucho through the FRPW motif. The ElB/Noc complex may thus serve to recruit the Groucho protein to specific target sites on the DNA, and repress the expression of distinct genes. Future identification of target genes will determine if ElB/Noc can also facilitate induction of certain genes (Dorfman, 2002).
Expression of ElB is confined to distinct tracheal branches from stage 12, namely the dorsal branch and lateral trunks. This restricted expression is instructive for the future fate and migration pattern of these branches. When misexpressed in other branches, ElB abolishes the migration of the visceral branch and eliminates the expression of a visceral branch marker. It also represses expression of Sal, a protein that defines the dorsal trunk identity. ElB is also able to divert several cells from the dorsal trunk fate into a dorsal branch (Dorfman, 2002).
In the tracheal branches where elB is normally expressed, elB has an essential role. In elB null mutants, fewer cells join the dorsal branch, and these branches migrate abnormally. In addition, the cells normally forming the lateral trunk anterior remain in the transverse connective, and the ganglionic branches are stalled. Attempts can be made to interpret these phenotypes with the repressive activity of the ElB/Noc complex in mind (Dorfman, 2002).
It is possible that in the dorsal branch, the complex represses expression of genes that confer dorsal trunk identity such as sal. A similar paradigm has been shown for the Dpp pathway. kni expression in the dorsal branch is induced by the Dpp pathway. Kni can bind the sal promoter and repress expression of the gene. In elB mutants expression of dorsal trunk genes extends to the dorsal branch and partially converts the identity of these cells to a dorsal trunk fate. Similarly, in the lateral trunk the ElB/Noc complex may be required to repress the expression of genes conferring visceral branch identity. In elB mutants, the expanded expression of visceral branch- and dorsal trunk-specific genes into the lateral trunk may thus abolish or stall the migration of the lateral trunk and ganglionic branches. Subsequently, ElB expression is confined to a specific cell within the dorsal branch, namely the fusion cell. In noc mutants the expression of Srf in the dorsal branch expands to the fusion cell. One way to interpret the expanded expression of Srf is by loss of direct repressive activity of ElB/Noc in the fusion cell (Dorfman, 2002).
Why is the elB mutant tracheal phenotype more severe than that of noc, if the two proteins form a functional complex? Since noc does not have an early maternal RNA, the zygotic noc mutant phenotype should reflect the null situation. ElB can associate not only with Noc, but also with another ElB monomer. It is thus possible that in noc mutant embryos, ElB/ElB homodimers partially substitute for the ElB/Noc heterodimers (Dorfman, 2002).
The tracheal defects observed in elB mutants are reminiscent of tracheal defects in tkv mutant embryos, where signaling of the Dpp pathway is blocked. However, ElB appears to function in parallel to the Dpp pathway. Phosphorylation of Mad and induction of kni expression, both of which mark the activity of the Dpp pathway, are normal in elB mutants. Conversely, ElB expression is independent of Dpp/Tkv activation. The possibility that Dpp signaling directs a post-translational modification of ElB/Noc has not been ruled out. Nevertheless, the available data suggests that generation of the dorsal branch, and migration of the lateral trunk anterior and ganglionic branch, require both the input from Dpp signaling and the expression of ElB/Noc (Dorfman, 2002).
It is not known yet how activation of distinct tracheal cells in the dorsal and ventral region of the tracheal pit by Dpp, as visualized by pMad accumulation, contributes to the capacity of these cells to form the dorsal and lateral branches, respectively. Activation by Dpp induces expression of target genes such as kni in these compartments. Kni in turn was shown to repress expression of dorsal-trunk genes like sal (Dorfman, 2002).
How are the ElB/Noc and Dpp signals integrated in the trachea? One possibility is that they impinge on different target genes. ElB/Noc repress expression of visceral branch or dorsal trunk genes, while the Dpp signal induces the expression of target genes in the same cells. The combined activity of the two pathways will determine the set of branch-specific genes expressed by these cells. The final identity of each branch is likely to be a result of inputs from different pathways, which contribute to the expression of branch-specific genes and to the repression of other genes. This is exemplified most clearly when monitoring kni expression in the dorsal branch of elB mutant embryos. While the correct number of cells are induced by the Dpp pathway and express kni, some of these cells are stalled in the dorsal trunk in the absence of ElB. It is also demonstrated by the fact that only the combined activity of ElB and activated Tkv is capable of inducing an excess of LTa cells. ElB/Noc and Kni also cooperate in the repression of common target genes. Complete repression of sal in the dorsal branch cells requires both complexes, as evidenced by the expanded expression of Sal in elB-mutant embryos (Dorfman, 2002).
Future knowledge regarding the nature of these branch-specific target genes should provide insights into the mechanism that regulate branch-specific fates. These genes may encode adhesion molecules or membrane receptors that allow responses to different sets of external guiding cues. This system could provide further migrational specificity, superimposed on the common Branchless signal guiding the migration of all tracheal branches. Furthermore, it may determine the stereotyped number of cells recruited into each tracheal branch (Dorfman, 2002).
The similarity in the phenotypes of elB and noc mutants, the genetic interaction between the mutants, and the complex formed between the two proteins, strongly suggest that these proteins carry out their biological roles as a complex. However, the two genes are regulated differently in the embryo. noc is broadly expressed and overexpression of the protein does not give rise to a tracheal phenotype, suggesting that spatial and temporal regulation of activity relies on elB expression. The restricted expression of elB is essential, since elB misexpression gives rise to deleterious phenotypes in the trachea. It is not known if additional tiers of regulation, such as inputs from signaling pathways or phosphorylation, also impinge on the complex (Dorfman, 2002).
Expression of elB is initially observed in all tracheal cells, suggesting that it is under the control of Trachealess and Drifter, which confer tracheal identity. However, at stage 12, expression of elB becomes excluded from the central part of the pit. It is possible that the restricted pattern of elB expression is thus a combination of induction by general tracheal transcription factors, and repression of expression in the future dorsal trunk and visceral branch. The signals leading to this repression are not known. The EGF receptor pathway is activated in the central domain of the tracheal placodes. However, when the activity of this pathway is abolished in Star mutants, elB expression remains unchanged (Dorfman, 2002).
It will be interesting to find out the function the ElB/Noc complex in other tissues. In noc mutant embryos, defects in migration of cells from the procephalic lobe are observed (Cheah, 1994). Expression of elB is not restricted to the trachea, and is also observed in the wing imaginal disc and the adult photoreceptors (S. Cohen and C. Desplan, communication to Drofman, 2002). In accordance with the roles of the ElB/Noc complex in the trachea, it is likely that in the above tissues the same complex will be essential for determination of cell fates, by repressing and possibly also inducing critical target genes (Dorfman, 2002).
In conclusion, using a tracheal misexpression screen two proteins have been identified that form a complex and participate in the determination of specific tracheal branch fates. ElB/Noc define a parallel input to Dpp signaling, demonstrating that convergence of several signals contributes to the robust determination of branch-specific cell fates, and to the refinement of these fates (Dorfman, 2002).
Plasmid rescue of the EP(2)2039 element shows that it is inserted at chromosomal position 35B, previously known as the elbow-noc (elB-noc) region (Davis, 1990; Davis, 1997; Ashburner, 1999). The EP line is inserted 965 bp upstream of the 5' UTR and 1365 bp upstream of the translation start site of elB, and is homozygous viable. Since no publicly available ESTs were found, an embryonic cDNA library was screened with the EP(2)2039 genomic rescue fragment. Three positive clones were sequenced and analyzed, all of them were 2550 bp and encoded a protein of 553 amino acids. The cDNA translation shows that there is a different exon-intron structure compared with both the published GeneScan predicted gene, and with CG4220 in the GadFly genomic annotation (Dorfman, 2002).
BLAST analysis of the cDNA sequence was performed and shows that ElB belongs to the Sp1 transcription factor family. However, it contains a single zinc finger (C2H2 type), while Sp1 transcription factors generally possess several zinc fingers. Antibody staining of ElB in embryos overexpressing elB indeed shows a nuclear localization pattern. The closest homolog of ElB is the Drosophila Noc protein, which shares 50% identity (Cheah, 1994). It has previously been reported that Noc contains two zinc fingers, but the algorithms used detected only one zinc finger. The noc gene is located in the same chromosomal region at a distance of 82 kb from elB, suggesting that it arose by a gene duplication event (Dorfman, 2002).
A human protein termed AK024361, which is localized to 8p11.2, and a putative transcript from chromosome 10 both show a high degree of similarity to ElB and Noc proteins. Alignment of these four proteins allowed identification of additional conserved domains. In the N terminus there is a putative activation domain, while a proline- and tyrosine-rich domain is located in the C terminus. In addition, there is a cysteine-rich domain in the middle of ElB protein. Between the activation domain and Cysteine-rich domain are several stretches of Alanine repeats. Previous studies have suggested that Noc functions as a repressor of transcription due to the high amount of the A-repeats (Cheah, 1994). A putative FKPY Groucho-binding motif is also found in all four proteins (Dorfman, 2002).
date revised: 20 October 2002
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