Regulation of adult female germline stem cells by nutrient-responsive signaling

Journal Pre-proof Regulation of Adult Female Germline Stem Cells by Nutrient-responsive Signaling Kun-Yang Lin, Hwei-Jan Hsu

PII:

S2214-5745(20)30001-8

DOI:

https://doi.org/10.1016/j.cois.2019.10.005

Reference:

COIS 646

To appear in:

Current Opinion in Insect Science

Please cite this article as: Lin K-Yang, Hsu H-Jan, Regulation of Adult Female Germline Stem Cells by Nutrient-responsive Signaling, Current Opinion in Insect Science (2020), doi: https://doi.org/10.1016/j.cois.2019.10.005

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Regulation of Adult Female Germline Stem Cells by Nutrient-responsive Signaling Kun-Yang Lin1, 2, 4 and Hwei-Jan Hsu1, 3, 4, ¶ 1

Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate

Program, National Chung Hsing University and Academia Sinica, Taipei 11529; 2Graduate

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Institute of Biotechnology, National Chung Hsing University, Taichung 40227; 3

Biotechnology Center, National Chung Hsing University, Taichung 40227; 4Institute of

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Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan ¶

Corresponding author: [email protected]; phone: 886-2-2787-1541; fax:

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886-2-27858059

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Highlights 1. Systemic factor, like Insulin/IGF signaling, controls dietary response of GSCs via acting on the GSC itself, GSC niche cells, and the adipose tissue surrounding the ovary. 2. The adipose tissue, a nutrient sensing organ, uses insulin/IGF signaling and amino acid response pathways to control GSC maintenance. 3. GSC intrinsic regulators, niche local signals, and adipocyte local signals are coordinated to control GSC response to nutrient inputs.

Abstract

Insect oogenesis is greatly affected by nutrient availability. When nutrients are abundant, oocytes are rapidly generated, but the process is slowed to 1

conserve energy under nutrient-deficient conditions. To properly allocate limited resources toward oogenesis, systemic factors coordinate the behavioral response of ovarian germline stem cells (GSCs) to nutritional inputs by acting on the GSC itself, GSC supporting cells (the niche), or the adipose tissue surrounding the ovary. In this review, we describe current knowledge of the Drosophila ovarian

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GSC-niche-adipocyte system and major nutrient sensing pathways (insulin/IGF signaling, TOR signaling, and GCN2-dependent amino acid sensing) that

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intrinsically or extrinsically regulate GSC responses to nutrient signals.

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Keywords: Insulin signaling, Notch, TOR, Dilp, Amino acid response pathway,

Introduction

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ecdysone, GSC

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Oogenesis in insects is typically nutrient-dependent, meaning it is triggered only

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when sufficient nutrients are available [1]. For example, in anautogenous female mosquitos, synthesis of vitellogenin (yolk protein for vitellogenesis) is increased in the fat body after ingestion of a blood meal [2]. Moreover, Drosophila on a protein-rich diet (yeast-diet) lay approximately 80 eggs per day, while those on a protein-poor diet (only water and sugar, which can be considered starvation conditions) 2

for only one day exhibit low vitellogenesis and slowed division of germline stem cell (GSCs) [3], the progenitors for making oocytes [4]. Importantly, this process is reversible, with egg production completely restored after switching flies from a protein-poor to a protein-rich diet for one day [3]. Thus, insects (especially Drosophila) clearly have mechanisms to rapidly regulate ovarian GSCs in response to

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nutrients. Compared to other insects, the ease of culture, well-known cell biology of the

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ovary, and availability of genetic tools make Drosophila an excellent model with which to study the effects of nutrient signals on ovarian GSCs [5]. In Drosophila

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females, 16-20 ovarioles comprise an ovary, with two or three GSCs located in the

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anterior-most structure of each ovariole, the germarium. Each GSC directly contacts

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two or three cap cells (CpCs) and anterior escort cells (ECs) (Fig. 1), which together form the GSC niche that houses and maintains GSCs [6]. Cap cells are considered the

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major niche component, as they provide Dpp stemness signals (orthologous to

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mammalian BMPs) [7] and E-cadherin-mediated physical contact to maintain GSC identity and residence [8]. Upon Dpp binding to Thickveins (Tkv) on the GSC, Mothers against Dpp (Mad) is phosphorylated by activated Tkv, and then signaling is initiated to suppress transcription of differentiation genes and maintain GSC cell fate [9]. Meanwhile, asymmetric division of GSCs generates differentiated daughter cells, 3

which later develop into mature oocytes/eggs [4]. Nutrients from digested food are absorbed by the small intestine and distributed to different organs via the hemolymph. Adipose tissue serves as both an energy reservoir and an endocrine organ, producing bioactive compounds that influence multiple processes, such as metabolic homeostasis and reproduction [10]. In insects,

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the fat body (composed of adipocytes and hepatocyte-like oenocytes) underneath the cuticle surrounds major organs, including brain and ovary [11]. Recent studies have

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shown that Drosophila adipocytes remotely control ovarian GSC maintenance [12-14], although the detailed mechanisms are still being delineated.

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It is clear that ovarian GSCs are tightly regulated by systemic factors acting on

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GSCs, niche cells, and adipocytes at multiple levels in order to coordinate GSC

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behavior with the nutritional status of the organism. Here, we summarize recent findings on how major nutrient-sensing pathways regulate Drosophila ovarian GSCs

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(Fig. 1), including results on insulin/insulin-like growth factor (IGF), Target of

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rapamycin (TOR), and amino acid response (AAR) pathways. Given the widespread evolutionary conservation of these pathways, these findings may be applicable to other insects, animals, and stem cell systems. Insulin/IGF signaling controls female GSC response to nutrient signals The insulin/IGF pathway is a highly conserved nutrient-sensing pathway [15], 4

wherein binding of insulin/IGFs to the Insulin receptor (InR) stimulates the receptor (known as Chico in fly) to activate phosphoinositide 3-kinase (PI3K) [16]. PI3K activity then recruits serine/threonine kinase Akt (known as Akt1 in fly) to the membrane, where it is phosphorylated and activated [16]. Akt subsequently modulates a variety of cellular processes via phosphorylation of its many substrates, such as the

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inhibitory phosphorylation of Forkhead Box O (Foxo; a transcription factor that negatively regulates insulin/IGF signaling) and Glycogen synthase kinase-3β

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(GSK-3β) [17]. Akt also phosphorylates the tuberous sclerosis complex (TSC)1/2,

leading to activation of TOR, which further activates Ribosomal protein S6 kinase

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(S6K) to promote protein synthesis [17]. As a master nutrient sensor, insulin/IGF

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signaling acts on GSCs, niche cells and adipocytes to modulate reproductive function

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via GSC proliferation and maintenance.

-Insulin/IGF signaling in GSCs controls GSC division

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Drosophila insulin-like peptides (Dilps), which are mainly produced in brain

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neuroendocrine cells, are received by GSCs and stimulate cell division [18]. Inr mutant GSCs display reduced proliferation with slowdown at G2, while the double mutation of chico and foxo partially rescues GSC division [19]. Interestingly, flies on a protein-poor diet exhibit GSC cell cycle delays at both G1 and G2 phases, suggesting that some unknown factors also regulate G1 progression in response to 5

diet. Although TOR signaling is involved in amino acid sensing [20], it is also activated by insulin/IGF signaling to regulate various cellular functions [21]. However, Tor acts independently of insulin signaling to cell-autonomously influence GSC proliferation and maintenance [22]. Flies carrying mutations in tor (ortholog of TOR)

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exhibit GSC cell cycle delay at G2, while G2 delay is not reversed in foxo and tor double-mutant flies [22]. In addition, GSCs with tor mutation are lost from the niche

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two weeks earlier than inr mutant GSCs [22-24]. Intriguingly, mutations of tsc1 or

tsc2, which encode Tor suppressors, cause severe GSC loss via hyperactivation of Tor

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critical for GSC maintenance.

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and impaired Dpp stemness signaling [22, 25]. Thus, a proper level of Tor signaling is

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-Insulin/IGF signaling in niche cap cells controls niche integrity for GSC maintenance via Notch

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Like inr mutants, a protein-poor diet also causes depletion of GSCs and niche

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cap cells [23]. However, GSCs with inr homozygous mutation induced by mitotic recombination can stay in the niche for two weeks [18, 26], while directly knocking down inr expression in the niche results in quick GSC loss, within a week (Su and Hsu, unpublished results). Therefore, insulin/IGF signaling in cap cells seems to maintain niche cell number, which in turn maintains GSCs. Mechanistically, 6

insulin/IGF signaling promotes E-cadherin expression in cap cell-GSC junctions for GSC maintenance [23], and supports cap cell survival via the Notch signaling pathway. Maturation of Notch receptor requires multiple sugar modifications, including the Fringe-mediated addition of N-acetylglucosamine [27]. Under a protein-poor diet, low insulin/IGF signaling in niche cap cells activates FOXO, which

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directly promotes Fringe expression at transcriptional level to block Notch signaling [28]. Fringe probably blocks Notch by increasing cis-inhibition between Notch and its

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ligands [29], thereby reducing Notch signaling in cap cells.

-Insulin/IGF signaling promotes membrane extension of niche escort cells for GSC

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maintenance

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Escort cells display long cellular protrusions that wrap germ cells to promote

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maturation [30]. Moreover, anterior escort cells directly contact GSCs to serve as part of the niche [31]. Importantly, these niche escort cells are also targets for insulin/IGF

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signaling to maintain GSCs [32]**. inr mutant or starved flies exhibit poorly wrapped

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germ cells/GSCs; however, re-feeding or overexpression of insulin/IGF signaling components in escort cells of starved flies rescues this defect. The membrane extension of escort cells is in part mediated by Failed axon connections (Fax), a diet-regulated membrane protein known to control neurite outgrowth [33, 34]. Fax translation is promoted by S6K activation, downstream of insulin/IGF signaling [32]. 7

Knocking down inr or fax, or overexpressing dominant-negative S6K in escort cells results in shortened membrane extensions, GSC loss, and attenuated Dpp stemness signaling, as revealed by the reduced expression of phosphorylated Mad in GSCs without effects on Dpp production. Interestingly, the reduction of Dpp stemness signaling was not observed in GSCs carried by inr mutant flies, as examined by the

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expression of daughter against dpp (dad)-lacZ [23], a Dpp signaling reporter [35]. These differing results could be due to a difference in the sensitivities of tools used for

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analyzing Dpp signaling; otherwise, the discrepancy could reflect different levels of

residual insulin signaling in the niche of inr mutant flies and the inr-knockdown niche.

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Nevertheless, these findings suggest that insulin/IGF signaling in niche escort cells

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promotes cytoskeleton rearrangement that facilitates Dpp delivery to GSCs.

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-Insulin/IGF signaling in adipocytes controls GSC maintenance via Akt1/GSK3 A recent study revealed that insulin/IGF signaling in adult adipocytes controls

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ovarian GSC maintenance [13]**. Blocking insulin signaling by knocking down inr

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or Akt1 in adipocytes reduces adipocyte size and diminishes the number of GSCs without affecting Dpp signaling or E-cadherin expression, suggesting other mechanisms mediate this GSC loss. Overexpression of Shaggy (GSK3 ortholog), but not Foxo, in adipocytes rescues GSC number, indicating that insulin/IGF signaling in adipocytes maintains GSCs via the Akt1/GSK3 regulatory axis [13]. These results 8

suggest that close communication occurs between adipocytes and the ovary, but the underlying mechanisms are as yet undetermined. The amino acid shortage sensor GCN2 in adipocytes disrupts GSC maintenance Amino acid sufficiency in adipocytes maintains GSCs by suppressing activation of General Control Non-depressible protein 2 (GCN2) [14], a major regulator of the

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AAR pathway [36]. Upon amino acid shortage, uncharged transfer RNAs (tRNAs) activate GCN2 to phosphorylate the translation initiation factor, eIF-2α, thereby

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inhibiting protein translation [37]. Knocking down amino acid transporters, including CG12773, slimfast, CG7708, CG1607, CG1628 and CG13384, in adipocytes causes

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GSC loss without affecting niche cap cell number, an effect that can be rescued by

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co-knockdown of GCN2 [14]. It remains unclear how GCN2-dependent AAR

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signaling in adipocytes negatively regulates GSC maintenance. Other factors may sense nutrients to control GSCs

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- Ecdysone signaling and Adiponectin Receptor (AdipoR) in GSCs control GSCs

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The steroid hormone, ecdysone, is synthesized from dietary cholesterol [38]. In adult flies, the production of ecdysone in old ovarian follicle cells is dependent on insulin and nutrient availability [39, 40]. Moreover, ecdysone signaling in GSCs regulates their division at G2/M phase [41], and it maintains GSCs via chromatin remodeling factors (e.g., ISWI and Nurf301) [41] that are known to affect Dpp 9

stemness signaling [42]. However, direct evidence of diet-linked GSC maintenance via ecdysone signaling is still lacking. Adiponectin, a diet-regulated protein hormone in mammals, maintains lipid homeostasis by promoting fatty acid oxidation and glucose consumption [43]. Although there is no homolog of Adiponectin in Drosophila, signaling via the

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Adiponectin Receptor (AdipoR) is required for GSC maintenance independent of insulin/IGF signaling [44]; impaired BMP signaling and E-cadherin-mediated

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niche-GSC cell adhesion partially mediate this effect [44]. Thus, lipid homeostasis may also play a role in GSC maintenance.

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in adipocytes control GSC maintenance

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-Fatty acid oxidation, phosphatidylethanolamine synthesis, iron transport and Upd2

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Adipose tissue can act as an endocrine organ, producing various bioactive compounds with widespread effects [45]. Proteomic analysis of fat tissue revealed

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that flies on a protein-poor diet exhibit deficiencies in fatty acid oxidation,

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phosphatidylethanolamine synthesis and iron transport pathways [12]**. Furthermore, knocking down any of several genes involved in these pathways – including CG3961 (fatty acid oxidation), easily shocked (phosphatidylethanolamine synthesis) and Ferritin 1 heavy chain homologue (iron transport) – within adipocytes causes GSC loss [12]. Furthermore, sugar and lipids were shown to promote adipocyte expression 10

of Unpaired 2 (Upd2), a leptin-like cytokine, that stimulates secretion of brain-derived Dilps [46]. Thus, mounting evidence shows that adipose tissue acts as a nutrient-sensing organ that impacts GSC maintenance. Insulin and steroid signaling pathways control Drosophila male GSCs in response to nutrients

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Like those in females, GSCs and their supporting cells in males are also affected by nutritional status. Feeding male flies with a protein-poor diet causes GSC

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proliferation to slow down [47], at least in part as a result of increased rates of

centrosome misorientation [48]. Protein starvation also reduces the numbers of both

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male GSCs and cyst stem cells [49, 50], which directly contact GSCs and acts as a

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component of the GSC [51]. The cyst stem cells help regulate GSC maintenance, and

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their progeny wrap GSC descendants to promote spermatogenesis [52]. Notably, these process are reversible and have been shown to be mediated by insulin signaling [49,

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50]. In contrast to ovarian GSCs, male GSCs require intrinsic insulin signaling for

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their maintenance and proper centrosome orientation [47, 48]. Constitutive expression of inr in early germ cells partially rescues the GSC reduction and centrosome misorientation induced by poor diet [48]. Moreover, expression of inr in both GSCs and hub cells, another component of the male GSC niche [51], nearly completely restores GSC number under starvation conditions [47], suggesting an important role 11

for insulin signaling in hub cells as well. Despite these intriguing observations, the specific roles of insulin signaling in the GSC niche are not fully understood. In males, autonomous ecdysone signaling is required for maintenance of cyst stem cells, and non-autonomous signaling controls GSCs [53]. Blocking ecdysone signaling in GSCs or hub cells does not change GSC number; however, disruption of

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ecdysone signaling in somatic stem cells reduces their number. Similar to the state of knowledge about ovarian GSCs, it is still unclear whether ecdysone signaling is

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involved in dietary regulation of male GSC behavior. Thus, while there are indications that nutrient signaling affects male GSC maintenance, the mechanisms of this

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Conclusions and open questions

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regulation have not been clearly defined in the male system.

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Systemic, local and intrinsic factors all participate in the multi-layered dietary responses of tissues, niche cells and GSCs to properly coordinate GSC function and

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nutritional status. In this review, we have summarized recent findings that elucidate

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the mechanisms of communication between niche cells, adipocytes, endocrine cells and GSCs via systemic factors, like insulin/IGFs and ecdysone. Among these intercellular communication events, adipocyte-GSC exchanges are

the least well understood. In adipocytes, manipulation of insulin/IGF signaling, AAR and metabolic pathways all result in GSC loss, suggesting that some secreted factor(s) 12

from adipocytes regulates GSCs, possibly targeting the ovary via effects on the brain or other organs. Future studies to identify this secreted factor(s) and the targeted organs will help to clarify how adipocytes remotely control GSCs in response to diet. It has also been reported that lipid metabolism in GSCs controls their maintenance [54]. Therefore, in addition to amino acid transporters and Tor-dependent

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amino acid sensing, metabolism in GSCs may also determine the GSC response to nutrient availability. A potential issue raised by this suggestion is that most studies on

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diet and GSCs use sugar and water as a protein-poor diet; however, protein is not the only missing nutrient (e.g., vitamins and lipids) in this diet. Detailed dissection of

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nutrient requirements for certain phenotypes would precisely reveal how GSCs are

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regulated by diet. Furthermore, mutations affecting GSC response to nutrients may act

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by decreasing Dpp stemness signaling and delaying G2/M progression. However, the mechanisms underlying these effects are still unclear. Further studies will illuminate

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how systemic factors mediate organ communication to control stem cells in response

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to nutritional inputs and how diets (or nutrients) affect stem cell metabolism.

Conflict of Interest The authors declare no competing financial interests.

Acknowledgments 13

We thank Marcus Calkins for English editing. Author contributions: K.-Y. Lin and H.-J. Hsu wrote the paper. The work was supported by intramural funding from the Institute of Cellular and Organismic Biology, Academia Sinica, and the Ministry of

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Science and Technology, Taiwan (107-2311-B-001-004-MY3).

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Figure legends Figure 1. Nutrient signals act on multiple cell types to coordinate proper GSC response to diet. GSCs directly contact niche cap cells (CpCs) and escort cells (ECs), while the ovary is surrounded by adipocytes. CpCs produce Dpp signals and E-cadherin (E-cad), both of which maintain GSCs. Dpp binds to Thickveins (Tkv),

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triggering phosphorylation of Mad (pMad) to promote stemness signaling in the GSC. After digestion of food in the gut, nutrients, such as sugar, lipids, cholesterol and

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amino acids, are taken up and delivered to different organs. Under nutrient-rich

conditions, Drosophila-like peptide (Dilp) production is increased, predominantly in

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brain neuroendocrine cells. Dilp binding to Insulin receptor (InR) stimulates

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insulin/IGF signaling in multiple cell types. In GSCs, Dilp/InR promotes GSC

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division. In CpCs, Dilp/InR promotes Notch (N) signaling for CpC survival, which in turn, maintains GSCs. In ECs, Dilp/InR promotes membrane extension for efficient

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delivery of Dpp, and in adipocytes, insulin/IGF signaling is also necessary to maintain

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GSCs. Cholesterol is a precursor of ecdysone steroids. Upon ecdysone binding to Ecdysone receptor (EcR), signaling is stimulated in GSCs to control GSC division and maintenance; in ECs, ecdysone signaling is also necessary to maintain GSCs. Additionally, Adiponectin Receptor (AdipoR) in GSCs controls GSC maintenance. In conditions of amino acid deficiency, GCN2, a major regulator of the amino acid 21

response (AAR) pathway, is upregulated by unloaded tRNAs in adipocytes to cause GSC loss. Functional fatty acid (FA) oxidation, phosphatidylethanolamine (PE) synthesis and iron transport pathways in adipocytes are also necessary to maintain GSCs. Unpaired2 (Upd2) is increased in adipocytes to promote the secretion of

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brain-produced Dilps. Fng, Fringe; TSC, tuberous sclerosis complex.

#12 Matsuoka S, Armstrong AR, Sampson LL, Laws KM, Drummond-Barbosa D. Adipocyte Metabolic Pathways Regulated by Diet Control the Female Germline Stem

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Cell Lineage in Drosophila melanogaster. Genetics. 2017;206(2):953-71. doi: 10.1534/genetics.117.201921. PubMed PMID: 28396508; PubMed Central PMCID: PMCPMC5499197. **This study reveals comprehensive metabolic networks in adipocytes in response to starvation. Authors also document that pathways of fatty acid oxidation, PE synthesis and iron transport in adipocytes remotely control GSC maintenance. #13 Armstrong AR, Drummond-Barbosa D. Insulin signaling acts in adult adipocytes via GSK-3beta and independently of FOXO to control Drosophila female germline

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stem cell numbers. Dev Biol. 2018;440(1):31-9. Epub 2018/05/08. doi: 10.1016/j.ydbio.2018.04.028. PubMed PMID: 29729259; PubMed Central PMCID: PMCPMC5988998. **This study shows that the single nutrient-sensing insulin/IGF signaling in adipocytes regulates multiple stages of the ovarian GSC lineage via different mechanisms, all of which are independent of FOXO. Instead, adipocytes utilize the insulin/IGF-Akt1-GSK3β regulatory axis to maintain GSCs; meanwhile, adipocyte Akt1 but not GSK3βcontrols early germline cyst survival, and an Akt-independent pathway in adipocytes regulates vitellogenesis. #32 Su YH, Rastegri E, Kao SH, Lai CM, Lin KY, Liao HY, et al. Diet regulates membrane extension and survival of niche escort cells for germline homeostasis via insulin signaling. Development. 2018;145(7). Epub 2018/03/20. doi: 10.1242/dev.159186. PubMed PMID: 29549109. **This study shows that in response to a protein-rich diet, insulin/IGF signaling promotes membrane extension of anterior escort cells, one of the components of GSC niche. This action maintains GSCs by facilitating the distribution of Dpp stemness 22

signals. In addition, membrane extension of posterior escort cells is also promoted by insulin/IGF signaling to control survival and proliferation of early germ cells. These results indicate that nutrient signals are able to module cell cytoskeleton to control cellular processes. Fig. 1. Nutrient signals act on multiple cell types to ensure GSC response to diet

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