Notch and Egfr signaling act antagonistically to regulate germ-line

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  • Notch and Egfr signaling act antagonistically toregulate germ-line stem cell niche formation inDrosophila male embryonic gonadsYu Kitadatea,b and Satoru Kobayashia,b,1

    aOkazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, Higashiyama, Myodaiji, Okazaki 444-8787,Japan; and bDepartment of Basic Biology, School of Life Science, Graduate University for Advanced Studies, Nishigonaka, Myodaiji, Okazaki 444-8585, Japan

    Edited* by Allan C. Spradling, Carnegie Institution of Science, Baltimore, MD, and approved June 22, 2010 (received for review March 18, 2010)

    Germ-line stem cells (GSCs) are maintained by the somatic micro-environment, or GSC niche, which ensures that GSCs can bothself-renew and produce functional gametes. However, it remainsunclear how the proper niche size and location are regulatedwithin the developing gonads. In the Drosophila testis, the hubcells that form the GSC niche are derived from a subset of somaticgonadal precursors (SGPs) in the anterior portion of the embryonicgonad. Here we show that Notch signaling induces hub differen-tiation. Notch is activated in almost all SGPs in the male embryo-nic gonad, but Epidermal growth factor receptor (Egfr) is activatedin posterior SGPs to repress hub differentiation, thereby restrictingthe expansion of hub differentiation in the embryonic gonad.We further show that Egfr is activated in posterior SGPs by Spitzligand secreted from primordial germ cells (PGCs), whereas theNotch ligand Serrate is expressed in SGPs. This suggests that vary-ing the number of PGCs alters niche size. Indeed, a decrease in thenumber of PGCs causes ectopic hub differentiation, which conse-quently increases their opportunity to recruit PGCs as GSCs. Whenectopic hub differentiation is repressed, the decreased number ofPGCs fails to become GSCs. Thus, we propose that SGPs sense PGCnumber via signals from PGCs to SGPs that modulate niche size,and that this serves as a mechanism for securing GSCs.

    primordial germ cell | pole cell | somatic gonadal precursor | hub cell

    Stem cells contribute to a steady source of new cells to maintaintissue homeostasis. Many stem cells reside in local microen-vironments, or niches, that act as sources of local extrinsic signalsto stem cells to maintain their self-renewing potential. Recently,niches have been identified in various tissues, such as theDrosophilaintestinal epithelium (13), ovary (4), and testis (5, 6), as well as themammalian skin epidermis (7), hematopoietic system (810), andneural tissues (1113). To maintain proper tissue architecture, thenumber of stem cells and the proper placement of their niche ina tissue are crucial. For example, misregulation of niche formationresults in tissue degeneration or tumor-like morphological abnor-malities (8, 9, 1418). Although the role of niches in the mainte-nance of stem cells and tissue homeostasis has been well-studied,relatively little is known about how niche size and location areprecisely regulated during development.One of the best-characterized stem cell niches is found in the

    Drosophila testis, in which easily identifiable germ-line stem cells(GSCs) and their niche cells maintain the continuous productionof sperm. At the apical tip of the testis, GSCs lie in intimatecontact with somatic niche cells, or hub cells, which send the self-renewal signal Unpaired (Upd), a ligand that activates Jak/STATsignaling pathway in the neighboring GSCs (5, 6, 19). Whena GSC divides, the daughter remaining in contact with the hubcells maintains stem cell identity, while the daughter that is dis-placed from the hub receives a weaker signal and initiates sper-matogenesis.GSCs are derived from pole cells that form in the posterior pole

    of early embryos (20). They migrate into the interior of the em-bryo and associate with somatic gonadal precursors (SGPs) to

    form the embryonic gonad, then become primordial germ cells(PGCs). At this early stage, the embryonic gonad is already sex-ually dimorphic (21, 22). In male embryos, hub cells are specifiedfrom the anterior SGPs at the end of embryogenesis (23, 24).These hub cells send a short-range signal, Upd, to the neighboringPGCs to recruit them as GSCs, whereas PGCs that do not asso-ciate with hub cells undergo spermatogenesis (24). In contrast, infemales, GSCs and their niche are formed later in third-instarlarvae (25, 26).We have previously reported that the receptor tyrosine kinase

    (RTK) Sev is required to ensure that the niche develops in theanterior region of the male embryonic gonads (17). Sev is ex-pressed in the posterior SGPs and is activated by the Boss ligandemanating from PGCs to prevent ectopic niche differentiation inthe posterior SGPs. However, Sev is not sufficient to repress hubdifferentiation in the anterior gonad (17), suggesting that anothersignaling pathway has a central role in restricting hub differenti-ation to the anterior SGPs. These findings also suggest that bothposterior and anterior SGPs have the capacity to contribute tohub differentiation, but the mechanisms by which they do thisremain elusive.In this study, we demonstrate that Notch and Epidermal growth

    factor receptor (Egfr) signaling act antagonistically to regulate hubdifferentiation in themale embryonic gonad.We show thatNotch isactivated in almost all SGPs to induce hub differentiation. Notch isactivated by Ser ligand emanating from SGPs. In contrast, activa-tion of Egfr in posterior SGPs by Spitz ligand secreted from PGCsis necessary and sufficient to repress hub differentiation. Thus,anterior and posterior SGPs become competent to differentiateinto hub cells through Notch-mediated interactions between SGPs,whereas PGCs signal to SGPs through Egfr and Sev to restrict hubdifferentiation to anterior SGPs. We further show that a decreasein the number of PGCs enhances ectopic hub formation, which inturn increases the recruitment of PGCs as GSCs. Thus, regula-tory interactions between PGCs and SGPs ensure that the propernumber of GSCs is generated within the male gonads.

    Results and DiscussionNotch Signaling Pathway Induces Hub Differentiation in the MaleEmbryonic Gonad. We tested the role of Notch signaling in hubdifferentiation within the male embryonic gonad, because Notchsignaling pathway is required for stem cell maintenance andniche formation in various systems (3, 16, 26, 27). Notch expres-sion was detectable in almost all SGPs from stage 12 until at leastthe end of embryogenesis in the male embryonic gonad (Fig. 1A).

    Author contributions: Y.K. and S.K. designed research; Y.K. performed research; Y.K.analyzed data; and Y.K. and S.K. wrote the paper.

    The authors declare no conflict of interest.

    *This Direct Submission article had a prearranged editor.1To whom correspondence should be addressed. E-mail:

    This article contains supporting information online at PNAS | August 10, 2010 | vol. 107 | no. 32 | 1424114246






  • To determine whether Notch is required for hub differentiation,we examined the expression of Fasciclin 3 (Fas3) inNotchmutantembryos. Fas3 is expressed in hub cells from embryogenesis untiladulthood (23, 28), and therefore is a good marker for hub cellidentity (17). In wild-type embryos, Fas3-positive SGPs were ob-served only in the anterior region of the male embryonic gonadfrom stage 15 onward (Fig. 1F). Notch264-39 and Notch5419 nullmutant gonads had a normal number of SGPs, and their overallmorphology was indistinguishable from wild type during stage1316 (Fig. 1 G and M). However, they began to degenerate atstage 17. In Notch264-39 mutant embryonic gonads, hub differen-tiation was severely impaired, with the number of Fas3-positivecells reduced compared with wild type (Fig. 1 G and M). Con-versely, when Notch activity was up-regulated in SGPs throughoutthe gonad by expression of the Notch intracellular domain(NotchICD), ectopic Fas3-positive cells were observed in the pos-terior of the gonad, and the number of Fas3-positive cells wassignificantly increased (Fig. 1 H and M). Based on these obser-vations, we conclude that Notch is necessary and sufficient toinduce hub differentiation throughout themale embryonic gonad.Because anterior SGPs contribute to hub differentiation dur-

    ing normal embryogenesis, we expected that Notch would beactivated only in the anterior SGPs. To detect Notch activation,we expressed a NotchICD-GAL4 transgene under the control ofa heat-shock promoter (hsp70-N-GV3) to activate a UAS-GFPreporter construct (29). Unexpectedly, GFP was detected in al-most all SGPs in the embryonic gonad (Fig. 1B), indicating that

    Notch is active in both the posterior and anterior SGPs of themale embryonic gonad.It has been reported that Notch is activated by binding to

    ligands encoded by Delta (Dl) or Serrate (Ser) (30). We detectedSer RNA in SGPs throughout the male embryonic gonad duringstage 1416 (Fig. 1C), whereas Dl-lacZ expression was unde-tectable (Fig. 1D). Consistent with these observations, the num-ber of Fas3-positive cells was significantly decreased in Ser null(SerRX106 and Ser94C) mutant embryos, compared with control(Fig. 1 I, J, and M), whereas Dl mutations did not affect hub dif-ferentiation (Fig. S1 A and B). These results suggest that SGPsproduce Ser ligand to activate Notch. This is further supported byseveral lines of evidence. First, hub differentiation occurs even inthe absence of PGCs (17, 31). Second, forced expression of Ser i