The similarity between ElB and No ocelli (Noc) proteins prompted an examination of the role of Noc in tracheal development. In situ hybridization shows that noc is expressed from stage 4 in the embryonic termini, and at stage 5/6 in ectodermal stripes. At stage 11, noc is expressed in the invaginating tracheal pits, while from stage 13, noc is expressed ubiquitously (Cheah, 1994). Since noc expression is not spatially restricted, it is not surprising that no tracheal phenotypes were observed after Noc misexpression by btl-Gal4/EP2000. Higher levels of induction of noc expression (using UAS-noc), do not abolish the visceral branches, but their direction of migration is occasionally misrouted. In addition, misexpression of both ElB and Noc in the trachea does not generate a phenotype that is more severe than the one produced by misexpression of ElB alone (Dorfman, 2002).
In order to examine the normal role of Noc in tracheal development, imprecise excisions were generated for the EP(2)2173 element, located 518 bp upstream of the 5'UTR. A lethal excision termed nocd64 was characterized by Southern blotting and inverse PCR. It represents a deletion of 848 bp, removing all residues between the insertion site of EP2173 and noc transcription start site, as well as 330 bp of the 5'UTR, thus defining a null mutation in noc. Homozygous nocd64 mutants show a weaker mutant phenotype, but similar to that of elB in terms of the affected tracheal branches. Fewer cells are observed in the lateral anterior trunk and ganglionic branch, and lateral branch fusion is occasionally missing. In addition, while all dorsal branches are formed, some branches have only up to three cells. The basis for partial lack of branch fusion in noc mutants was examined. Normally, in the dorsal branch only one terminal cell expresses SRF, while the preceding cell becomes the fusion cell and expresses specific markers. In noc mutants, repression of SRF expression in the fusion cell fails, giving rise to duplicated terminal cells at the expense of fusion cells (Dorfman, 2002).
In view of the overlapping tracheal expression patterns and the previously reported genetic interactions (Davis, 1997), it is possible that the ElB and Noc proteins form a heterodimeric complex. Since each protein contains only a single zinc finger, a heterodimer may provide the appropriate number of zinc fingers for DNA binding. GST pull-down experiments were carried out using GST fusions to full-length constructs of the ElB protein, as well as to fragments of the ElB protein. Association with in vitro translated full-length ElB or Noc was examined. Indeed, GST-ElB is capable of associating with Noc. In addition, GST-ElB is also capable of associating with ElB, demonstrating that ElB can form homodimers as well as heterodimers with Noc. Truncated constructs of ElB show that its C-terminal region, which contains the Cysteine-rich and zinc-finger domains, is sufficient for these interactions (Dorfman, 2002).
The biological activities of ElB and Noc are consistent with repression of transcription. Overexpression of ElB in the trachea represses the expression of specific genes in the dorsal trunk and visceral branch. Conversely, absence of ElB results in expanded expression of Sal to the dorsal branch, and noc mutants display failure to repress SRF expression in the fusion cells of the dorsal branch. The notion that the complex may posses repressive activity by virtue of its association with known inhibitors of transcription was tested. Two such proteins are known: CtBP and Groucho (Dorfman, 2002).
The Groucho protein is known to mediate long-range transcriptional repression, and to associate with DNA-binding proteins bearing a number of motifs, including FKPY. This sequence is conserved in ElB, Noc and the two human homologs. Thus the capacity of ElB to associate with Groucho was tested. Indeed, specific association was detected. CtBP promotes short-range repression and is known to associate with DNA-binding proteins containing the PxDLSxR/K/H motif. Such a motif is not found in ElB or Noc, and the GST-ElB fusion protein shows only negligible precipitation of labeled CtBP. This result strongly suggests that the ElB/Noc complex represses transcription of target genes directly, by recruiting Groucho to these sites (Dorfman, 2002).
In situ hybridization with an elB RNA probe reveals expression of elB in all tracheal pit cells, starting at stage 11. There is a higher level in the first and the tenth pits. At stage 12, as the primary branches form, elB expression is reduced within the central part of the pit, and becomes restricted to the lateral anterior and posterior branches and to the dorsal branch cells. The elB probe is specific and does not crossreact with noc RNA, which exhibits a different distribution (Dorfman, 2002).
Anti-ElB antibodies are able to detect ectopically expressed protein, but marginally detect the endogenous protein levels. However, a Gal4 element inserted 1260 bp upstream of the 5' UTR provides a sensitive expression pattern marker that correlates with the RNA pattern. A Gal4-responsive UAS-elB element shows that at stage 14 elB expression is detected in the lateral branches and excluded from the dorsal trunk and visceral branch. By stage 16 expression in the transverse connective, spiracular branch, lateral and ganglionic branches is observed. Within the dorsal branch, ElB is specifically expressed at this stage in the fusion cell. Restriction of ElB expression is indeed necessary, since expression of ElB in all branches is sufficient to eliminate the visceral branch, and leads to defects in the dorsal trunk (Dorfman, 2002).
In a misexpression screen of the EP collection with the midline- and tracheal-specific btl-Gal4 driver, the EP(2)2039 line gave rise to a lethal phenotype. Examination of the tracheal system of these embryos with antibodies recognizing the tracheal lumen revealed a specific abolishment of the visceral branch. By immunohistochemical analysis using anti-Trh antibody that detects all tracheal nuclei, as well as anti-ElB antibody, which shows a similar pattern, it was observed that cells that normally form the visceral branch remain in the central region of the transverse connective branch. In the dorsal branch, an increase in the number of cells was identified. Quantitation of the number of dorsal branch cells have demonstrated that while in wild-type embryos each branch contains an average of five cells, in ElB misexpression embryos most dorsal branches contain five to eight cells. The phenotypes of ElB tracheal misexpression were verified by crossing the btl-Gal4 line to lines bearing a pUAST construct driving an elB full-length cDNA. Similar phenotypes of reduced visceral branches and enlarged dorsal branches were observed (Dorfman, 2002).
To obtain a finer resolution, molecular markers for the different branches were followed. By using the l(2)01351 enhancer trap line that marks the visceral branch cells, it was shown that this marker is not expressed in the cells remaining in the transverse connective following ElB misexpression. Overexpression indeed abolishes expression of a gene normally marking the visceral branch cell fate. knirps (kni) is normally expressed in the lateral trunks (Lta and LTp), dorsal branch and visceral branch. Uniform expression of ElB in the trachea does not alter the pattern of kni expression in cells that usually form the LT. kni expression is also observed in visceral branch cells that are retained in the transverse connective upon ElB misexpression. Thus, not all visceral branch markers are abolished. Finally, in the dorsal branch more cells express kni due to ectopic migration of cells at the expense of the dorsal trunk (Dorfman, 2002).
In ElB misexpression embryos, several cells normally assigned to the dorsal trunk appear to be migrating into the dorsal branch, thus increasing the cell number in that branch. This suggests that there may also be a defect in specifying the dorsal trunk fate. Therefore, a dorsal trunk marker, Spalt (Sal), was followed in embryos misexpressing ElB. Sal is a transcription factor that is specifically expressed in the dorsal trunk cells and determines their identity. Ectopic ElB expression abolishes all Sal expression in the trachea. Surprisingly, this does not lead to an absence of the dorsal trunk, which is typical of sal mutant embryos, presumably because the btl-Gal4 driver induces the accumulation of ElB and abolishment of Sal expression only after execution of the normal Sal function in the dorsal trunk (Dorfman, 2002).
These experiments suggest that ElB expression must be excluded from the dorsal trunk and visceral branch, in order to specify proper dorsal trunk and visceral identities (Dorfman, 2002).
To determine the role of ElB in the branches where it is normally expressed, analysis of mutations in the elB locus was carried out. Several mutant fly lines in the elB region are available; however, they have not been precisely mapped. In order to create a line with a specific elB mutation, the EP element was excised, using a Delta 2-3 transposase. Only one lethal excision line (termed elBd47) showed rearrangements in Southern blot analysis in the region of EP2039. Inverse PCR of the rearranged genomic fragment and sequencing provided the following molecular details. The EP2039 element was deleted. All sequences between EP2039 insertion site and elB were intact, while on the other side sequences of the transposable yoyo element were identified. Further Southern analysis indicated a deletion of the genomic sequence in the region upstream of EP2039 insertion. Therefore, it is possible that an inversion coupled to a deletion of sequences upstream of EP2039 has taken place. elBd47 is lethal over Df(2L)noc10 and over the smaller deficiency Df(2L)fn2, uncovering 35A3-35B2. While elBd47 does not remove genomic regions containing the elB transcribed region, it is nevertheless allelic to the EMS-induced viable mutation elB1, which displays the typical smaller wing phenotype. It is thus possible that crucial transcriptional regulatory sequences of elB are removed in elBd47 (Dorfman, 2002).
Homozygous elBd47 embryos display defects in tracheal development. In all segments, there is no migration of the lateral trunk anterior, and the ganglionic branches are shorter. The cells that fail to migrate into the lateral trunk remain in the transverse connective. The number of cells that form the dorsal trunk and visceral branch do not change. Dorsal branch migration is also impaired, and failure of dorsal branch fusion is observed at stage 16. Cell counts of dorsal branch cells in the mutant embryos show a considerable number of branches with only three to four cells. In some of the segments, the dorsal branch cells migrate aberrantly, and laterally adjacent branches fuse. The same tracheal phenotype is observed in embryos homozygous for the Df(2L)fn2 deficiency, as well as trans-heterozygotes of elBd47 over Df(2L)fn2, demonstrating that elBd47 is a null mutation (Dorfman, 2002).
Since elBd47 may not affect only the elB gene, it was necessary to confirm that the observed tracheal phenotypes indeed result from loss of elB. Btl-Gal4 was used to drive UAS-elB inserted on the third chromosome, in the background of homozygous elBd47 embryos. The most pronounced tracheal phenotype of elBd47 embryos is manifested in stalled anterior lateral trunk migration. Since tracheal misexpression of elB does not give rise to defects in the LTa of wild-type embryos, it was possible to examine rescue of this phenotype. Embryos misexpressing ElB were identified by anti-ElB staining and the characteristic visceral branch defects. While 25% of these embryos are homozygous for elBd47, no severe LTa migration defects were observed. It was possible to identify specifically some of the rescued homozygous elBd47 embryos, by virtue of subtle defects in other branches such as the dorsal and ganglionic branch (Dorfman, 2002).
The tracheal phenotype of elB mutants is reminiscent of defects arising when signaling by the Dpp pathway is blocked (e.g. in thickveins mutant embryos). Most notable is the stalled migration of the lateral trunk anterior, the ganglionic branch and the dorsal branch. Dpp is expressed at stage 11 in two ectodermal stripes: the dorsal stripe is restricted to a single row of the dorsal-most ectodermal cells, and is positioned several cells away from the dorsal edge of the tracheal pit. The ventrolateral stripe of Dpp expression abuts the ventral side of the tracheal pit (Dorfman, 2002).
Activation of the Tkv/Punt receptors by Dpp leads to the phosphorylation of the Mad protein, which forms a heterodimer with Medea and translocates to the nucleus to trigger transcription. Antibodies specifically recognizing the C-terminal phosphorylated form of Smad1 also recognize the phosphorylated form of Drosophila Mad. These antibodies were used to follow Dpp signaling in the trachea in wild-type and elB mutant embryos (Dorfman, 2002).
In wild-type embryos, pMad is observed in the tracheal pits beginning at early stage 11, when Dpp is expressed in two stripes in the dorsal and ventrolateral ectoderm. Dpp activation subdivides the tracheal pit into three parts: the activated dorsal and ventral domains and the central region, which is not activated by Dpp. The number of cells in which pMad is generated in the ventral domain of the pit is significantly larger than the number of tracheal cells displaying pMad in the dorsal domain. Since the dorsal stripe of Dpp is positioned several cell diameters above the tracheal pit, the diffusion of Dpp reaches and activates only approx. five tracheal cells. This corresponds to the number of cells that will be recruited to the dorsal branch (Dorfman, 2002).
The ventral Dpp stripe abuts the tracheal pit, and pMad is observed in more cells in the ventral aspect of the pit, in accordance with the larger number of lateral trunk cells influenced by Dpp signaling. It is interesting to note that despite the higher levels of Tkv expression in the tracheal pits, the amount of pMad in the tracheal cells versus the adjacent ectodermal cells is comparable. As migration of the tracheal branches continues, the lateral trunk cells migrate ventrally beyond the stripe of Dpp. Consequently, only the tracheal cells lying under this stripe retain pMad activation (Dorfman, 2002).
The pMad patterns allow a direct determination of whether the reception of Dpp signaling is compromised in elB mutants. elBd47 embryos were stained with the pMad antibody, and identified by the stalled migration of the lateral trunk anterior. The number of dorsal and ventral cells displaying pMad in the pit at stage 11/12 is comparable with wild-type embryos. ElB is thus not required for normal signaling by the Dpp pathway (Dorfman, 2002).
ElB could be required downstream of pMad, for the induction of Dpp-target genes. Expression of kni, which encodes a zinc-finger transcription factor, is induced by the activated Tkv/Punt receptors. kni-lacZ transgene was used to follow the dependence of kni expression on ElB. Overexpression of ElB does not alter the expression pattern of kni. Likewise, in elB mutants, the expected number of dorsal branch cells retain kni expression. However, while normally the kni-expressing cells segregate completely from the dorsal trunk into the dorsal branch, in the elB mutant several cells are retained within the dorsal trunk. This may be a reflection of failure of the cells destined to form the dorsal branch to lose their identity completely as dorsal trunk cells. Indeed, when expression of the dorsal trunk marker Sal was examined, residual Sal was observed in all the kni-expressing cells that remained within the dorsal trunk, and in some of the cells forming the dorsal branch. Thus, Kni is not sufficient to repress Sal expression, and requires cooperation with ElB for complete repression (Dorfman, 2002).
The normal reception of Dpp signaling and kni expression in elB mutants still leaves open the possibility that ElB is a downstream component of the Dpp pathway. Whether expression of elB is regulated by the Dpp pathway was also tested. Overexpression of activated Tkv with the btl-Gal4 driver does not alter the expression pattern of elB RNA. In addition, ectopic kni or knrl does not alter the elB expression pattern in the trachea, in agreement with the above results with activated Tkv. These results imply that while ElB and the Dpp pathway affect the same tracheal branches, they function in a parallel manner (Dorfman, 2002).
The combined effects of ElB misexpression and uniform activation of Tkv in the trachea could be manifested in cell fate changes leading to excess LTa cells. Following misexpression of only ElB or activated Tkv, the number of LTa cells is unchanged, while the typical visceral branch and dorsal branch abnormalities are observed. However, misexpression of both constructs also leads to changes in LTa cell fates, as extra cells are recruited into this branch (Dorfman, 2002).
The elbow locus is found to be two genes elA and elB, each of which has a distinct phenotype when mutant. Mutations of the elA gene have a strong phenotype where the wing is markedly disrupted. Mutations of elB are weak, mainly affecting the alula and the wing bristles. The two genes are dominant enhancers of each other. Homozygous deletion of the complete elbow region results in lethality. Situated between the elbow genes is the pupal gene and a locus which when deleted causes a crippled leg phenotype. This locus may be a control region for elbow. Immediately adjacent on the proximal side of elA is the no-ocelli locus. The phenotypes of noc alleles vary from extreme, where the ocelli and associated bristles are absent, to weak, where these structures are disrupted. The various noc phenotypes are associated with genetically distinct gene regions, mutations of which act as enhancers of each other. Alleles of el and noc show partial failure of complementation, heterozygotes having weak el or weak noc phenotypes. Alleles of both these genes interact with the antimorphic noc allele Sco (Davis, 1997).
The el-noc complex spans a distance of about 200 kb on chromosome 2L. It consists of three discrete genetic regions el, l(2)35Ba and noc, each of which has a distinct phenotype when mutant. The noc locus itself is complex, including three separate regions. The el locus has been characterized by mapping 30 aberration breakpoints to the DNA. It extends over a distance of about 80 kb. It can be divided into two parts by the aberrations In(2LR)DTD128 and T(Y;2)A80. These break between two sets of el alleles yet are both phenotypically wild type for elbow. The simplest explanation is that el consists of two transcription units elA and elB. The locus pu, which appears to be unrelated to the el-noc complex, is found to map between the two el loci very close to elB (the distal el locus). The loci l(2)35Ba and nocA have been separated by only two l(2)35Ba+nocA- deletions and a nocA- inversion. No l(2)35Ba-nocA+ aberrations have been found. At the molecular level these loci are found to occupy almost the same region, and are probably identical (Davis, 1990).
The phenotype for mutations of the nubbin gene consists of a severe wing size reduction and pattern alterations, such as transformations of distal elements into proximal ones. nub expression is restricted to the wing pouch cells in wing discs from the early stages of larval development. These effects are also observed in genetic mosaics where cell proliferation is reduced in all wing blade regions autonomously, and transformation into proximal elements is observed in distal clones. Mutant clones are approximately 50% smaller than control clones or else they fail to grow in 50% of the cases. Clones located in the proximal region of the wing blade cause an additional nonautonomous reduction of the whole wing. Cell lineage experiments in a nub mutant background show that clones respect neither the anterior-posterior nor the dorsal-ventral boundary but that the selector genes decapentaplegic and engrailed are correctly expressed from early larval development. The phenotypes of nub elbow and nub dpp genetic combinations are synergistic and the overexpression of dpp in clones in nub wings does not result in overproliferation of the surrounding wild-type cells (Cifuentes, 1997).
A screened was carried out for genes that, when overexpressed in the proliferating cells of the eye imaginal disc, result in a reduction in the size of the adult eye. After crossing the collection of 2296 EP lines to the ey-GAL4 driver, 46 lines were identified, corresponding to insertions in 32 different loci, that elicit a small eye phenotype. These lines were classified further by testing for an effect in postmitotic cells using the sev-GAL4 driver, by testing for an effect in the wing using en-GAL4, and by testing for the ability of overexpression of cycE to rescue the small eye phenotype. EP lines identified in the screen encompass known regulators of eye development including hh and dpp, known genes that have not been studied previously with respect to eye development, as well as 19 novel ORFs. Lines with insertions near INCENP, elB, and CG11518 were characterized in more detail with respect to changes in growth, cell-cycle phasing, and doubling times that were elicited by overexpression. RNAi-induced phenotypes were also analyzed in SL2 cells. Thus overexpression screens can be combined with RNAi experiments to identify and characterize new regulators of growth and cell proliferation (Tseng, 2002).
The lines EP965 and EP2039 have insertions that are 171 bp apart and in the same orientation, ~16.7 kb upstream of the gene elB (CG4220). The EP2039 insertion has been shown to be a weak elB allele, and a putative open reading frame (BG:DS06238.3) was identified by the Berkeley Drosophila Genome Project. This gene is predicted to encode a zinc-finger protein and shows 27% homology to no-ocelli (noc), a gene 100 kb proximal to BG:DS06238.3. The function of elB is unknown. No other transcription units have been identified between the EP insertions and elB. Both EP965 and EP2039 expression increased levels of elB RNA in a GAL4-dependent manner (Tseng, 2002).
The properties of proliferating cells were examined in the wing imaginal disc in the EP2039 line. Compared to the wild-type control, overexpression of the EP2039 line does not have any observable effects on cell size as determined by forward scatter. However, EP2039 overexpression does result in a slight change in cell-cycle phasing. There is a small but reproducible increase in the G2/M population, suggesting that EP2039 overexpression may be able to restrict either entry into or passage through mitosis. The population doubling time of cells overexpressing EP2039 was determined. Overexpression of EP2039 and p35 results in a doubling time of 18.6 hr, which is 26% longer than the doubling time of 14.8 hr calculated for the control cells expressing p35 alone. An increased doubling time with no change in cell size is consistent with a decreased rate of growth (mass accumulation) with a concomitant slowing of the cell cycle (Tseng, 2002).
Ashburner, M., Misra, S., Roote, J., Lewis, S. E., Blazej, R., Davis, T., Doyle, C., Galle, R., George, R. and Harris, N. (1999). An exploration of the sequence of a 2.9-Mb region of the genome of Drosophila melanogaster: the Adh region. Genetics 153: 179-219. 10471707
Cifuentes, F. J. and Garcia-Bellido, A. (1997). Proximo-distal specification in the wing disc of Drosophila by the nubbin gene. Proc. Natl. Acad. Sci. 94(21): 11405-11410.
Cheah, P. Y., Meng, Y. B., Yang, X., Kimbrell, D., Ashburner, M. and Chia, W. (1994). The Drosophila l(2)35Ba/nocA gene encodes a putative Zn finger protein involved in the development of the embryonic brain and the adult ocellar structures. Mol. Cell. Biol. 14: 1487-1499. 8289824
Davis, T., Trenear, J. and Ashburner, M. (1990). The molecular analysis of the el-noc complex of Drosophila melanogaster. Genetics 126: 105-119. 2121591
Davis, T., Ashburner, M., Johnson, G., Gubb, D. and Roote, J. (1997). Genetic and phenotypic analysis of the genes of the elbow-no-ocelli region of chromosome 2L of Drosophila melanogaster. Hereditas 126: 67-75. 9175495
Dorfman, R., et al. (2002). Elbow and Noc define a family of zinc finger proteins controlling morphogenesis of specific tracheal branches Development 129: 3585-3596. 12117809
Tseng, A.-S. K. and Hariharan, I. K. (2002). An overexpression screen in Drosophila for genes that restrict growth or cell-cycle progression in the developing eye. Genetics 162: 229-243. 12242236
Weihe, U., et al. (2004). Proximodistal subdivision of Drosophila legs and wings: The elbow-no ocelli gene complex. Development 131: 767-774. 14757638
date revised: 10 August 2004
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