stem cell tumor/rhomboid-2


DEVELOPMENTAL BIOLOGY

brho expression was surveyed during development by in situ hybridization in embryos, third-instar imaginal discs, and the female germ line. Expression is detected in early stage oocytes. brho expression was analyzed by performing PCR on various cDNA libraries and RT-PCR on RNA extracted from various developmental stages. Consistent with the very restricted expression of brho in the female germ line determined by in situ hybridization, a brho RNA product could be amplified from adult females by RT-PCR, but not from RNA extracted from embryos, larvae, pupae, or male adults. No brho transcripts were detected in cDNA libraries made from embryos or imaginal discs (Guichard, 2000).

Expression of brho in the female germ line is restricted to the early developing oocyte between stage 5 and stage 8. No brho staining was observed in the surrounding follicle cells, however. Egfr signaling has been reported to play at least three separate roles during early and late oogenesis: (1) to package a single oocyte and its attached nurse cells in the germarium prior to formation of the egg chamber; (2) to establish posterior follicle cell fates (stage 1-8 egg chambers), and (3) to establish the D/V axis in dorsal anterior follicle cells (stage 9-12 egg chambers). The latter two functions are required to pattern the egg chamber and embryo (Guichard, 2000).

The restriction of brho expression to the narrow window of oocyte development between stages 5 and 8 suggests that Brho might be involved in activating Egfr signaling in adjacent follicle cells required to specify posterior cell fates (Guichard, 2000).

stet transcript has been detected on Northern blots from adult testes, adult ovaries and 0-4 hour embryos. Transcript was not detected on similarly loaded Northern blots of mRNA from 4-24 hour embryos. Although the stet mutant phenotype clearly demonstrates a requirement for stet function in male germ cells, no stet mRNA was detected by in situ hybridization or stet protein by immunofluorescence staining of whole testes. The high load required to detect stet transcript on Northern blot and the failure to detect stet RNA or protein in whole mount testes suggests that stet is expressed at extremely low level. Similarly, although stet function is clearly required in region 1 and 2A of the germarium, neither stet mRNA nor stet protein accumulates at detectable levels at the tip of the germarium. stet mRNA was detected in germ cells in region 2B and 3 of the germarium. Signal from the stet mRNA is extremely low in germ cells of stage 1 and 2 egg chambers, but increases in germ cells of stage 3 to stage 8 egg chambers. In stage 1 to 8 egg chambers, stet mRNA appears to accumulate in the posterior region of the egg chambers, in the position of the developing oocyte (Schulz, 2002).

Effects of Mutation and Overrexpression

As a first step in assessing the function of brho, the GAL4-UAS system was used to misexpress a UAS-brho construct containing the full genomic sequence of brho in the wing. When this UAS-brho construct was expressed using the strong ubiquitous wing-specific GAL4 driver MS1096, ectopic vein phenotypes were observed similar to, although weaker than, those generated by misexpression of rho. This observation suggests that Brho functions like Rho by promoting Egfr signaling. To test whether the induction of ectopic veins by brho misexpression requires Egfr activity, UAS-brho was co-expressed with a dominant-negative Egfr construct, UAS-DN-Egfr. As previously observed for rho, brho-induced ectopic veins are entirely suppressed by DN-Egfr, resulting in narrow wings with missing veins typical of DN-Egfr misexpression. Strong synergism was observed between misexpressed brho and Star, as has been shown to be the case for rho. These results are consistent with brho functioning to promote Egfr signaling (Guichard, 2000).

Since Egfr signaling results in MAPK activation, the activation state of MAPK was assessed following misexpression of brho in the wing disc. MAPK is an essential downstream component required to transduce signals from all RTKs to the nucleus. Activated MAPK (MAPK*) can be detected in situ, using an antibody directed against phosphorylated MAPK (anti dP-ERK antibodies). In wild-type wing discs, MAPK activation is restricted to vein primordia, as a consequence of endogenous localized rho expression. In wing discs ubiquitously misexpressing brho, a strong general activation of MAPK was observed comparable to that found in discs ectopically expressing rho or an activated form of Egfr. This observation provides independent support for brho activating the Egfr/MAPK signaling pathway (Guichard, 2000).

As a direct measurement of Egfr activity during oogenesis, wild-type ovaries were probed with anti-dP-ERK antibodies. During early stages, MAPK activation is detected only in posterior follicle cells abutting the oocyte in which brho and gurken are expressed. This pattern of MAPK activation is temporally correlated with brho expression and is consistent with the hypothesis that brho participates in promoting Egfr signaling in posterior follicle cells. It is noteworthy that rho, which activates Egfr signaling in many other developmental settings, is not expressed in the oocyte or surrounding follicle cells during this period (Guichard, 2000).

During later stages of oogenesis (9-10), MAPK activation is restricted to follicle cells overlying the dorsal anterior end of the oocyte. This restricted activation of MAPK is believed to be the result of the asymmetrical localization of gurken transcripts to the dorsal anterior portion of the oocyte, which then resolves into a double peak as a consequence of rho, argos, and spitz activity in the dorsalmost anterior follicle cells at stage 11. Interestingly, a trace of posterior activation of MAPK is also observed at stage 10, suggesting that sustained posterior Egfr activity may maintain posterior fates of the egg chamber (Guichard, 2000).

Whether brho could activate the Egfr pathway in the ovary was tested by expressing the UAS-brho construct under the control of the CY2-GAL4 driver, which is expressed only in the follicular epithelium covering the oocyte. This ubiquitous follicle cell expression of brho causes dorsalization of the eggshell, resulting in thickened dorsal appendages which were more spread apart in eggs from CY2;UAS-brho females than in wildtype controls. In some more extreme cases, white appendage-like material filled in between the two appendages, as is typical of dorsalized eggshells. The average brho misexpression phenotype is similar to, but weaker than, that induced by ectopic rho in follicle cells using the same GAL4 driver (Guichard, 2000).

Star is expressed in the oocyte during a developmental window (stage 4 to 7) largely overlapping with brho expression. Since Star and rho act in concert during many stages of development and function in a strict interdependent fashion during wing vein development, tests were performed to see whether brho might also interact synergistically with Star. UAS-brho and UAS-Star constructs were coexpressed during wing development using the strong ubiquitous GAL4 driver MS1096, and highly penetrant pupal lethality was observed. Despite the pupal lethality, fully differentiated wings can be dissected from pupal cases, revealing a strong ectopic vein phenotype which is much greater than that observed in response to ectopic brho alone. Since ectopic expression of Star alone has no detectable effect, this result reveals a potent synergism between Brho and Star in enhancing Egfr activity during wing development (Guichard, 2000).

A strong effect on brho activity was observed from reducing the dose of endogenous Star since brho-induced ectopic veins are almost completely suppressed in a Star2/1 heterozygous background. These results indicate that Star can collaborate with brho, as well as with rho, to activate Egfr signaling (Guichard, 2000).

The data presented thus far suggest that the Brho protein can function early during oogenesis by activating Egfr signaling in follicle cells adjacent to the oocyte where brho is expressed. As a possible mechanism, it is proposed that Brho might promote processing or activation of the Grk protein in the oocyte to stimulate Egfr expressed in adjacent follicle cells. Consistent with the idea that mGrk, like mSpi, requires activation, the mGrk protein does not exhibit any activity when misexpressed in the wing. In contrast, an artificially truncated version of Grk, GrkDTM, can activate Egfr both in the wing and in follicle cells. In order to determine whether activation of mGrk involves Star, as has been observed for mSpi, Star and gurken were coexpressed during wing development (Guichard, 2000).

Coexpression of mgrk and Star results in a strong ectopic vein phenotype, which is greater than that caused by coexpression of mspi and Star. This finding supports the view that the Grk EGF ligand can be activated through a mechanism similar to that of mSpi (Guichard, 2000).

To determine whether Brho can also participate in activating Grk, UAS-brho and UAS-mgrk were co-expressed in the wing. The ectopic vein phenotype resulting from the coexpression of brho and mgrk is significantly stronger than that caused by brho alone, indicating that Brho can activate the mGrk precursor. A synergistic effect between brho and mspi was observed, similar to that which has been observed between rho and mspi . The phenotypes resulting from coexpressing brho + mgrk are significantly stronger than those from coexpressing brho + mspi; however, it is not believed that this necessarily reflects a preference of Brho for activating Grk versus Spi, since coexpression of UAS-Star with these ligands also results in a much stronger phenotype with Grk than Spi. These data suggest rather that the UAS-mGrk construct may be expressed more efficiently or at higher levels than the UAS-mspi construct. Also, it was not possible in these experiments to determine whether there was a significant increase in the severity of the phenotype resulting from coexpression of UAS-rho with either UAS-mspi or UAS-mgrk in the wing since UAS-rho generates a very strong ectopic vein phenotype when misexpressed alone (Guichard, 2000).

It is concluded that brho functions like rho by collaborating with Star in activating Egfr/MAPK signaling. Brho can potentiate the activity of both mSpi and mGrk EGF ligands, consistent with the possibility that Brho may activate Grk to promote Egfr/MAPK signaling and define posterior fates in the early follicular epithelium (Guichard, 2000).

Females mutant for loss-of-function stet alleles that cause severe defects in male germ cell differentiation produced few progeny (one to three adult progeny/female) and showed a variety of defects in oogenesis. In young stet mutant females, 60% of the ovarioles contained egg chambers at several different stages of differentiation. DAPI staining and phase contrast microscopy revealed that egg chambers from stet mutant females often contain abnormal numbers and arrangements of germ cells. In older stet mutant females, 90% of the ovarioles usually had only a few egg chambers, which commonly show signs of degeneration. By 2 weeks after hatching, ovarioles from stet mutant females are mostly empty, except for the germaria, which contained increased numbers of early germ cells. stet mutant females become completely sterile with increasing age (Schulz, 2002).

Early germ cells appeared to accumulate at the apical tip of the germarium in both young and old stet mutant females. In wild-type germaria, germline stem cells lie at the tip, followed by their immediate daughters, the cystoblasts, and then the interconnected cystocytes. In wild type, germline stem cells and cystoblasts can be distinguished from later stage germ cells by several subcellular markers. Sex-lethal (Sxl) protein accumulates in the cytoplasm of stem cells and cystoblasts to a much higher level than in later stage germ cells. In addition, alpha-spectrin is localized to the ball-shaped spectrosome in wild-type stem cells and cystoblasts but localizes to the branched fusome in cystocytes. Germaria from stet mutant females have an elevated number (ranging from the normal six to 75 cells) of early germ cells with cytoplasmic Sxl and a spectrosome compared with wild-type germaria (four to six cells). The apparent accumulation of cells resembling stem cells and/or cystoblasts in stet mutant germaria suggests that wild-type function of stet in females plays a role in allowing differentiation of early germ cells (Schulz, 2002).

Wild-type function of stet appears to facilitate the contacts between female germ cells and a population of somatic cells in region 1 and 2A of the germarium. In wild-type and stet mutant germaria, 11 to 12 inner sheath cells were detected in region 1 and 2A of the germarium based on the nuclear targeted lacZ enhancer trap marker I-72 for inner sheath cells. These inner sheath cells form cytoplasmic extensions between stem cells, cystoblasts and clusters of cystocytes in region 1 and 2A of the germarium, that can be seen at the ultrastructural level. The cytoplasmic extensions can also be seen upon expression of cytoplasmic GFP (UAS-GFP) under control of either an engrailed-GAL4 (en-GAL4) or a ptc-GAL4 transcriptional activator. In wild-type germaria, nine to 12 GFP-positive extensions were detected from inner sheath cells between germ cells in region 1 and 2A of the germarium. In germaria from stet mutant females, six to 12 GFP-positive inner sheath cells were present. However, they did not form normal numbers of cytoplasmic extensions. In 50% of the germaria from newly enclosed females, two to eight GFP-positive cytoplasmic extensions from inner sheath cells were detected around or between germ cells. By 1 week after hatching, no cytoplasmic extensions from inner sheath cells were detected in germaria from stet mutant females (Schulz, 2002).

Control of germline stem cell division frequency--a novel, developmentally regulated role for epidermal growth factor signaling

Exploring adult stem cell dynamics in normal and disease states is crucial to both better understanding their in vivo role and better realizing their therapeutic potential. This study addresses the division frequency of Germline Stem Cells (GSCs) in testes of Drosophila melanogaster. GSC division frequency is under genetic control of the highly conserved Epidermal Growth Factor (EGF) signaling pathway. When EGF signaling was attenuated, a two-fold increase in the percentage of GSCs in mitotic division was detected compared to GSCs in control animals. Ex vivo and in vivo experiments using a marker for cells in S-phase of the cell cycle showed that the GSCs in EGF mutant testes divide faster than GSCs in control testes. The increased mitotic activity of GSCs in EGF mutants was rescued by restoring EGF signaling in the GSCs, and reproduced in testes from animals with soma-depleted EGF-Receptor (EGFR). Interestingly, EGF attenuation specifically increased the GSC division frequency in adult testes, but not in larval testes. Furthermore, GSCs in testes with tumors resulting from the perturbation of other conserved signaling pathways divided at normal frequencies. It is concluded that EGF signaling from the GSCs to the CySCs normally regulates GSC division frequency. The EGF signaling pathway is bifurcated and acts differently in adult compared to larval testes. In addition, regulation of GSC division frequency is a specific role for EGF signaling as it is not affected in all tumor models. These data advance understanding concerning stem cell dynamics in normal tissues and in a tumor model (Parrott, 2012).

This study reports on the division dynamics of GSCs in response to attenuated EGF signaling. GSCs in EGF mutant testes contained more cells in M-phase and in S-phase of the cell cycle and it took significantly less time for all GSCs within one testes to complete one round of the cell cycle compared to GSCs in control testes. Confirming the role for EGF signaling in regulating the frequency of GSC divisions, germline-specific expression of EGF ligand rescued the hyperproliferation of GSCs in EGF mutant animals. Mutations in stet as well as RNAi-mediated knockdown of the EGFR in cyst cells recapitulated the increased mitotic activity of GSCs. These data demonstrate a novel and specific role for EGF signaling: the repression of GSC division frequency (Parrott, 2012).

Based on these findings, a model is proposed that demonstrates the bifurcation of the EGF signaling pathway (see A model depicting the requirement for EGF signaling). EGF acts in the CySCs in one pathway to regulate GSC division frequency and in a different pathway in cyst cells to promote germ cell enclosure and differentiation. This developmental bifurcation of EGF function during Drosophila spermatogenesis reveals a fundamental uncoupling between the control of stem cell proliferation and the control of stem cell daughter differentiation. The stage-specific requirement for EGF may reflect the different functions of GSCs in immature versus mature tissues. The initial function of GSCs may be to quickly populate larval testes with germline cells, while GSCs in adult testes need to replenish differentiated cells dependent on demand (Parrott, 2012).

This study is the first report of a stage-specific impact of a signaling pathway on the activity of GSCs and suggests that this developmental switch in GSC activity between larval and adult stages requires the activities of stage-specific pathways. On a molecular level, additional pathways may be active during larval stages that counteract the increased division frequency observed in adult GSCs upon loss of EGF. In larval testes, nutrient availability and cell growth may be the primary factors governing the frequency of GSC divisions. Conversely, soon after eclosion, Drosophila males reach sexual maturity and spermatogenesis may rely on EGF-mediated signaling to regulate GSC divisions (Parrott, 2012).


REFERENCES

Ghiglione, C., et al. (2002). Mechanism of activation of the Drosophila EGF Receptor by the TGFalpha ligand Gurken during oogenesis. Development 129: 175-186. 11782411

Guichard, A., Roark, M., Ronshaugen, M. and Bier, E. (2000). brother of rhomboid, a rhomboid-Related Gene expressed during early Drosophila oogenesis, promotes EGF-R/MAPK signaling. Dev. Biol. 226: 255-266. 11023685

Kiger, A. A., White-Cooper, H. and Fuller, M. T. (2000). Somatic support cells restrict germline stem cell self-renewal and promote differentiation. Nature 407: 750-754. 11048722

Parrott, B. B., Hudson, A., Brady, R. and Schulz, C. (2012). Control of germline stem cell division frequency--a novel, developmentally regulated role for epidermal growth factor signaling. PLoS One 7(5): e36460. PubMed Citation: 22586473

Pascall, J. C. and Brown, K. D. (1998). Characterization of a mammalian cDNA encoding a protein with high sequence similarity to the Drosophila regulatory protein Rhomboid. FEBS Lett. 429(3): 337-340. PubMed Citation: 9662444

Schulz, C., et al. (2002). Signaling from germ cells mediated by the rhomboid homolog stet organizes encapsulation by somatic support cells. Development 129: 4523-4534. 12223409

Urban, S., Lee, J. R. and Freeman, M. (2002). A family of Rhomboid intramembrane proteases activates all Drosophila membrane-tethered EGF ligands.EMBO J. 21: 4277-4286. 12169630

Wasserman, J. D., et al. (2000). A family of rhomboid-like genes: Drosophila rhomboid-1 and roughoid/rhomboid-3 cooperate to activate EGF receptor signaling. Genes Dev. 14: 1651-1663. PubMed Citation: 10887159


stem cell tumor/rhomboid-2: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 30 December 2012

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