Lilach Gilboa

Soma-germline interactions coordinate homeostasis and growth in the Drosophila gonad

The ability of organs such as the liver or the lymphoid system to maintain their original size or regain it after injury is well documented. However, little is known about how these organs sense that equilibrium is breached, and how they cease changing when homeostasis is reached. Similarly, it remains unclear how, during normal development, different cell types within an organ coordinate their growth. Here we show that during gonad development in the fruitfly Drosophila melanogaster the proliferation of primordial germ cells (PGCs) and survival of the somatic intermingled cells (ICs) that contact them are coordinated by means of a feedback mechanism composed of a positive signal and a negative signal. PGCs express the EGF receptor (EGFR) ligand Spitz, which is required for IC survival. In turn, ICs inhibit PGC proliferation. Thus, homeostasis and coordination of growth between soma and germ line in the larval ovary is achieved by using a sensor of PGC numbers (EGFR-mediated survival of ICs) coupled to a correction mechanism inhibiting PGC proliferation. This feedback loop ensures that sufficient numbers of PGCs exist to fill all the stem-cell niches that form at the end of larval development. We propose that similar feedback mechanisms might be generally used for coordinated growth, regeneration and homeostasis.

Germ line stem cell differentiation in Drosophila requires gap junctions and proceeds via an intermediate state

Gap junctions coordinate processes ranging from muscle contraction to ovarian follicle development. Here we show that the gap junction protein Zero population growth (Zpg) is required for germ cell differentiation in the Drosophila ovary. In the absence of Zpg the stem cell daughter destined to differentiate dies. The zpg phenotype is novel, and we used this phenotype to genetically dissect the process of stem cell maintenance and differentiation. Our findings suggest that germ line stem cells differentiate upon losing contact with their niche, that gap junction mediated cell-cell interactions are required for germ cell differentiation, and that in Drosophila germ line stem cell differentiation to a cystoblast is gradual.

How different is Venus from Mars? The genetics of germ-line stem cells in Drosophila females and males

In the fruit fly Drosophila melanogaster, both spermatogenesis and oogenesis rely on germ-line stem cells (GSCs). Intensive research has revealed many of the molecules and pathways that underlie GSC maintenance and differentiation in males and females. In this review, we discuss new studies that, some differences notwithstanding, highlight the similarities in the structural and molecular strategies used by the two sexes in GSC maintenance and differentiation. These include the tight control that somatic support cells exert on every aspect of GSC function and the similar molecular mechanisms for physical attachment, cell-cell signaling and gap-junction communication. Some common principles underlying GSC biology in the fly may be applied to stem cells in other organisms.

Coordinated regulation of niche and stem cell precursors by hormonal signaling.

In the developing Drosophila ovary, the ecdysone signaling pathway controls the differentiation of both niche and germ line stem cell precursors.

Insulin and Target of rapamycin signaling orchestrate the development of ovarian niche-stem cell units in Drosophila

Tissue-specific stem cells and their niches are organized into functional units that respond to external cues in order to maintain organ homeostasis. Insulin and Target of rapamycin (Tor) signaling mediate external cues that control adult niches and stem cells. Whether these pathways play a role in the establishment of niche-stem cell units during organogenesis has been little explored. We show that during larval development both Insulin-like receptor (InR) and Tor participate in the establishment of ovarian niches and germline stem cells (GSCs) in Drosophila melanogaster. Tor and InR are required cell-autonomously for the proliferation of precursors for both somatic niches and GSCs. These pathways also promote the formation of terminal filaments (part of the somatic niche). Significantly, InR, but not Tor, signaling non-autonomously promotes primordial germ cell (PGC) differentiation. Somatic attenuation of the pathway retards PGC differentiation, whereas its activation results in their precocious differentiation. We also show that InR-mediated PGC differentiation is independent of somatic ecdysone signaling, but that further differentiation into cysts requires an ecdysone input. These results demonstrate that Tor and InR signaling actively participate in the formation of ovarian niches and stem cells by affecting both cell numbers and differentiation. The dual influence of Tor and InR on both somatic cells and PGCs ensures that these two cell populations develop coordinately. Our work further identifies a novel step in the regulation of germ cell differentiation by demonstrating that following bag of marbles expression, cyst formation requires an additional hormonal input.

Without children is required for Stat-mediated zfh1 transcription and for germline stem cell differentiation

Tissue homeostasis is maintained by balancing stem cell self-renewal and differentiation. How surrounding cells support this process has not been entirely resolved. Here we show that the chromatin and telomere-binding factor Without children (Woc) is required for maintaining the association of escort cells (ECs) with germ cells in adult ovaries. This tight association is essential for germline stem cell (GSC) differentiation into cysts. Woc is also required in larval ovaries for the association of intermingled cells (ICs) with primordial germ cells. Reduction in the levels of two other proteins, Stat92E and its target Zfh1, produce phenotypes similar to woc in both larval and adult ovaries, suggesting a molecular connection between these three proteins. Antibody staining and RT-qPCR demonstrate that Zfh1 levels are increased in somatic cells that contact germ cells, and that Woc is required for a Stat92E-mediated upregulation of zfh1 transcription. Our results further demonstrate that overexpression of Zfh1 in ECs can rescue GSC differentiation in woc-deficient ovaries. Thus, Zfh1 is a major Woc target in ECs. Stat signalling in niche cells has been previously shown to maintain GSCs non-autonomously. We now show that Stat92E also promotes GSC differentiation. Our results highlight the Woc-Stat-Zfh1 module as promoting somatic encapsulation of germ cells throughout their development. Each somatic cell type can then provide the germline with the support it requires at that particular stage. Stat is thus a permissive factor, which explains its apparently opposite roles in GSC maintenance and differentiation.

Hormonal Control of Stem Cell Systems

Many organs respond to physiological challenges by changing tissue size or composition. Such changes may originate from tissue-specific stem cells and their supportive environment (niche). The endocrine system is a major effector and conveyor of physiological changes and as such could alter stem cell behavior in various ways. In this review, we examine how hormones affect stem cell biology in four different organs: the ovary, intestine, hematopoietic system, and mammary gland. Hormones control every stage of stem cell life, including establishment, expansion, maintenance, and differentiation. The effects can be cell autonomous or non-cell autonomous through the niche. Moreover, a single hormone can affect different stem cells in different ways or affect the same stem cell differently at various developmental times. The vast complexity and diversity of stem cell responses to hormonal cues allow hormones to coordinate the bodys reaction to physiological challenges.

Activin signaling balances proliferation and differentiation of ovarian niche precursors and enables adjustment of niche numbers

How the numbers of niches and resident stem cells within a particular organ are determined during development and how they may be modulated or corrected is a question with significant medical implications. In the larval ovary of Drosophila melanogaster, somatic precursors for niches, and germ cells that will become germline stem cells, co-develop. Somatic precursors proliferate during the first 3 days of larval development. By mid-third instar, adult terminal filament (TF) (part of the germline stem cell niche) cells first appear, and differentiation terminates 24 h later when 16-20 TFs fully form. The developmental sequence responsible for TF cell determination and final TF numbers is only partially understood. We show that TF formation proceeds through several, hitherto uncharacterized stages, which include an early exit from the cell cycle to form TF precursors and two steps of cell shape change to form the mature TF cells. The Activin receptor Baboon (Babo) is required for somatic precursor cell proliferation and therefore determines the pool of TF precursors available for TF differentiation. During the final differentiation stage, Babo facilitates TF and germ cell differentiation, and promotes the accumulation of Broad-Z1, which is also a target of the steroid hormone ecdysone. Epistasis analysis shows that Activin controls cell proliferation in an ecdysone-independent manner and TF differentiation by affecting ecdysone targets. We propose that this mode of function allows Activin to balance proliferation and differentiation, and to equilibrate niche numbers. These results suggest a novel model for how niche numbers are corrected during development. Summary: In the Drosophila ovary, terminal filament cell development is regulated by activin, which, along with the hormone ecdysone, controls expression of the key transcription factor Broad-Z1.

Organizing stem cell units in the Drosophila ovary

Organogenesis utilizes processes fundamental to development: cell proliferation, cell differentiation and morphogenesis. Each of these processes is complex in itself; the challenge of studying organogenesis is to determine how all of them integrate to shape organs with recurring precision. This review focuses on the emerging understanding of how synchronized proliferation and differentiation of both somatic and germ cell lineages form 16-20 germ line stem cell (GSC) units in the ovary of Drosophila melanogaster. Recent work demonstrates that the Insulin, ecdysone, Epidermal Growth Factor, Decapentaplegic and Activin signaling pathways are used reiteratively for proliferation and differentiation in both somatic and germ cell lineages. This linkage underlies ovarian coordinated development and provides opportunity for correction mechanisms for stem cell unit numbers.

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) (1, 2). The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches (3-12). Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar (13-17). GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs (7, 16, 18, 19). Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism. Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100μm across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.