Gene name - seven up
Cytological map position - 87B4--87B5
Function - transcription factor
Keywords - eye development
Symbol - svp
Genetic map position - 3-
Classification - orphan nuclear receptor
Cellular location - nuclear
The protein Seven-up is a so-called orphan nuclear receptor belonging to the steroid receptor superfamily that includes ligand dependent transcription factors such as the vertebrate glucocorticoid receptor, the thyroid hormone receptor and the retinoic acid receptor. Why does the term "orphan" nuclear receptor apply to SVP? For an answer, look to the interaction with ligand. For many of these transcription factors there is a cytoplasmic phase and a nuclear phase, induced by interaction with ligand, the small steroid molecules that trigger the activation of receptors. In the nuclear phase steroid receptors act as transcription factors. In the case of Seven-up, the ligand for its activation (if there is one) is currently unknown, and thus the appelation "orphan" nuclear receptor.
Seven-up is required for development of four of the eight photoreceptors that develop in each ommatidium of the eye, the R3/R4 pair and the R1/R6 pair. These photoreceptors are determined after R8 and the R2/R5 pair, but before the determination of R7. Two recent studies show the requirement for Ras signaling in seven-up function (Begemann, 1995 and Kramer, 1995). The Ras pathway is required in R7 cells to mediate between the Sevenless receptor and the proteins Pointed and Yan, but Sevenless signaling is not required for Seven-up function (Hiromi, 1993).
The activation of Ras pathway signaling on behalf of Seven-up seems to require the activity of Egf-r, the Drosophila homolog of the vertebrate epidermal growth factor receptor. Like Sevenless signaling, Egf-r activates the Ras pathway. A genetic trick has been used to provide evidence for the involvement of EGF-R. When seven-up is expressed in outer cone cells (the non-neuronal components of ommatidia), a neuronal transformation takes place, and ectopic R7 cells are induced. Suppressors of this transformation are mutants in EGF-R. This indicates that the neuronal transformation of cone cells requires EGF-R, and by inference, normal function of seven-up is similarly thought to require EGF-R (Begemann, 1995).
It is still not clear what role the Ras pathway plays in the activation of Seven-up function, nor is it clear how seven-up is activated in photoreceptor precursors. One identified target of Seven-up in cell determination is pipsqueak (Weber, 1995), but its role in cell fate determination is not well undersood. Because Seven-up is an orphan nuclear receptor, it would be of interest to see whether Seven-up function is tied to molting signals that are driven by other members of the nuclear receptor superfamily.
The Drosophila Malpighian tubules (MTs), form a simple excretory epithelium comparable in function to kidneys in vertebrates. MTs function as the insect kidney both in the larva and the adult. They consist of two pairs of blind ending tubes that are composed of a single cell-layered epithelium made up of a tightly controlled number of cells. The tubules float in the hemolymph from where they take up nitrogenous waste that is excreted as uric acid. During embryogenesis, MTs evert as four protuberances from the hindgut primordium, the proctodeum. The everting tubules grow by cell proliferation, which takes place in a few cells along the tubules and extensively in a distal proliferation domain located in the tip region of the tubules. Cell ablation experiments and studies on the pattern of cell division have shown that a single large cell at the distal end of each tubule, termed the tip cell, is decisive for controlling the proliferation of its neighboring cells. The tip cell that differentiates into a cell with neuronal characteristics during later stages of development arises by division of a tip mother cell that is selected in the tubule primordium by lateral inhibition involving the Notch signaling pathway and the transcription factor Kruppel (Kr). It has been suggested that the tip cell sends a mitogenic signal to adjacent cells in the distal proliferation zone. It has remained elusive, however, what the signal is or what its target molecules in the signal-receiving cells could be and how cell proliferation during MT morphogenesis is regulated. Seven-up is shown to be a key component that becomes induced in response to mitogenic EGF receptor signaling activity emanating from the tip cell. Seven-up (Svp) in turn is capable of regulating the transcription of cell cycle regulators (Kerber, 1998).
Two types of transcripts have been characterized at the svp locus: svp type I encodes a protein with both a DNA-binding domain and a ligand binding domain (LBD); and svp type II diverges from type I in the middle of the LBD. During MT development, both isoforms of svp are expressed in the same pattern. Their expression can first be detected in embryonic stage 10 on one side of the outgrowing tubules and, later, during the eversion, in a group of about six to eight cells in the tip region. Analysis of the MTs of amorphic svp mutants reveals a reduction of the tubule cell number, as compared to wild type. Anti-Kr antibody stainings reveal that the MT precursor cells are specified normally in svp mutants, indicating that the cause for the defect is not attributable to cell death, which might lead to a size reduction of the tubule primordium. Furthermore, tip cell determination occurs normally in the mutants. Rather, pulse labeling with BrdU, suggests that the reduction of the cell number results from a failure of proper cell divisions. In wild-type embryos, BrdU incorporation occurs asymmetrically on one side of each tubule in proliferating cells. When MT eversion begins in stage 10, the dividing cells in the distal tip region continue to incorporate BrdU extensively until the end of stage 13 when division stops. Subsequently, intense BrdU incorporation occurs in all of the tubule cells during endomitotic cycles that take place in a proximal to distal direction in the tubules. In svp mutants relatively normal BrDU incorporation is found during the initial cell divisions, but subsequently it is strongly reduced indicating a failure of DNA replication. In the later occurring endomitotic cycles, the BrdU pattern is normal again, indicating that a specific block of S phases occurs in dividing cells, but not during the endomitotic cycles. These results suggest that svp, which is expressed in the proliferation domains marked by BrdU, might be an integral component of the regulatory network that regulates division in the cells that receive the mitogenic signal from the tip cell (Kerber, 1998).
To identify the nature of the mitogenic tip cell signal a screen was carried out for genes specifically active in the tip cells. The genes rhomboid (rho) and Star (S), which encode transmembrane proteins involved in epidermal growth factor receptor (EGFR) signaling, are expressed in the tip cells and both are required for MT growth. When the tubules start to evert, rho and S are expressed in the tip mother cell; subsequently rho is strongly expressed in the tip cell and S in the tip cell and its former sister cell. An analysis of the MTs in the corresponding amorphic mutants reveals a strong decrease of cells in rho mutants and a weaker decrease in S mutants. In a rho;S double mutant, the tubules are barely detectable, indicating that rho and S activities are essential (albeit redundant) components controlling MT growth. The tubule phenotype of rho;S double mutants is very similar to that of EGFR mutants, which also show a drastic decrease in the tubule cell number. As in svp mutants, the allocation and the differentiation of the tip cells are normal in the receptor mutants, indicating that receptor activity is not required for tip cell determination and differentiation. The reduction of the tubule cell number in EGFR mutants is due to a failure of proper cell divisions. No BrdU incorporation occurs in EGFR mutants in the outbudding tubules at the time when cells divide in wild-type embryos. However, BrdU incorporation occurs again much later during the endomitotic cycles, indicating that in EGFR muants, a specific defect in DNA replication exists in cells that would normally divide (Kerber, 1998).
Rho and S process a membrane-bound form of the activating ligand of the receptor, the TGFalpha-like Spi protein, to generate the secreted form of Spi (sSpi). sSpi is then proposed to diffuse to neighboring cells, bind to the receptor, and activate target genes via the Ras/Raf signaling cassette; these include the primary target gene pointedP1 (pntP1), encoding an ETS domain transcription factor, and the secondary target gene argos (aos), encoding a negatively acting ligand of the receptor. These downstream components of the pathway are also active during tubule development. pntP1 and aos are expressed during stage 10 in six to eight cells on one side of the MTs overlapping the rho and S expression domains and later, weakly in several cells in the tip region. In amorphic aos mutants a slightly larger number of tubule cells are observed, whereas amorphic pnt mutants show a decrease of tubule cells. These results indicate that for controlling cell proliferation and cell determination, the same key components of the EGFR cascade are required (Kerber, 1998).
These findings suggest that the EGFR pathway provides the mitogenic tip cell signal that activates svp expression and regulates cell division. To test this hypothesis, svp expression was analyzed in EGFR mutants and ectopic expression studies were performed with various members of the pathway using the UAS-Gal4 system. svp is absent in mutants for the Egfr. It is still expressed, however, in amorphic pnt mutants, suggesting that Svp is a transcriptional regulator that is likely to be activated in parallel to the primary transcription factor PntP1 in the signaling cascade. If sSpi activity is provided ectopically in all of the tubule cells, the svp expression domain becomes dramatically expanded and an increase of the tubule cell number is observed. Similar, although slightly weaker effects on svp transcription and the number of tubule cells could be observed upon ubiquitous expression of other components of the EGFR pathway, like Rho, activated Ras, or Raf. Conversely, when a dominant-negative Ras allele is ectopically expressed in all of the tubule cells, svp transcription became strongly reduced. Ectopic expression of svp in an Egfr mutant background restores the tubule cell number to a considerable extent. These results provide strong evidence that svp is a downstream target gene of EGFR signaling in the tubules (Kerber, 1998).
If Svp is expressed ectopically in wild-type MTs, an increased number of tubule cells is obtained. BrdU incorporation studies indicate that this increased cell number results from extra cell divisions, indicating that svp is both necessary and sufficient to induce cell proliferation in the MTs. Analyses were carried out to further elucidate how the EGFR pathway and svp control cell proliferation, and whether these developmental regulators have an impact on components of the cell cycle machinery during MT growth. Two genes are limiting key components of the cell cycle during the period when the MT cells proliferate: string (stg), which encodes a Cdc25 phosphatase involved in the regulation of the G2/M transition, and cyclin E (cycE), which regulates the G1/S transition. In situ hybridization reveals that both genes are expressed asymmetrically in the everting tubules and subsequently in the distal proliferation zone. These expression domains match the svp expression domain. With the onset of the endomitotic cycles, a second phase of cycE expression occurs from proximal to distal in the tubules. In EGFR mutants, the transcriptional activation of stg and cycE, which occurs in the tubule proliferation domains in wild type, cannot be detected. This correlates with a strong reduction of BrdU incorporation and the dramatic reduction of the tubule cell number in Egfr mutants. During the subsequent endomitotic cycles, expression of cycE is not affected, indicating a specific function of EGFR signaling in activating early cycE expression. In svp mutants, the expression of stg and cycE is reduced (most likely reflecting that Svp is only one of the regulators that transmits the mitogenic EGFR signal); however, in MTs in which svp is ectopically expressed, stg becomes transcriptionally misexpressed in the cells that undergo extra cell divisions. Similar (although weaker) misexpression is obtained with cycE. However, extra cell divisions can only be obtained early during MT outgrowth, suggesting that other regulators limit cell proliferation during later stages of MT development (Kerber, 1998).
It is not known whether Svp, whose function has been characterized initially in the context of photoreceptor development in the eye also plays a role for cell proliferation during eye imaginal disc development. In MT there must be other factors in addition to Svp that are dependent on EGF signaling and are involved in MT growth. This is apparent from the finding that the svp mutant phenotype is less severe than that of Egfr mutants. Those predicted factors might include other steroid hormone receptors that interact with Svp as cofactors. Studies on ecdysone signaling pathways show that Svp can heterodimerize with subunits of the ecdysone receptor and regulate gene expression. Whether ecdysone-based signaling pathways also play a role in controlling cell proliferation in the MT is not known. Once cell proliferation is completed, the tubule cells elongate as a result of cell rearrangement and long thin tubes are generated with only two or three cells surrounding the lumen. An additional role of EGFR signaling during later stages of MT development cannot be excluded. This is consistent with recent results obtained with an antibody against the activated form of MAP kinase, which visualizes the activated state of receptor tyrosine kinase (RTK) signaling pathways and shows a rather uniform actived ERK pattern in all of the tubule cells. As there is no apparent tubule elongation defect in svp mutants, other downstream factors must be involved in mediating this potential aspect of EGFR signaling. In summary, these data provide a framework for further analysis of the molecular mechanisms that underlie the control of cell proliferation by developmental regulators during MT morphogenesis (Kerber, 1998).
There are two transcripts, types 1 and 2, differing in their 3' sequences. The transcripts are identical up to base number 1807, coding for amino acid number 452 (Mlodzik, 1990).
Bases in 3' UTR - 980 and 601 for transcripts type 1 and 2 respectively
date revised: 21 July 98
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