Cell non-autonomous function of daf-18/PTEN in the somatic gonad coordinates somatic gonad and germline development in C. elegans dauer larvae

Claudia C Tenen; Iva Greenwald

doi:10.1016/j.cub.2019.01.076

. Author manuscript; available in PMC: 2020 Mar 18.

Published in final edited form as: Curr Biol. 2019 Feb 28;29(6):1064–1072.e8. doi: 10.1016/j.cub.2019.01.076

Cell non-autonomous function of daf-18/PTEN in the somatic gonad coordinates somatic gonad and germline development in C. elegans dauer larvae

Claudia C Tenen ¹, Iva Greenwald ^2,³

PMCID: PMC6486834 NIHMSID: NIHMS1520543 PMID: 30827916

Summary

C. elegans larvae integrate environmental information and developmental decisions [1–3]. In favorable conditions, worms develop rapidly and continuously through four larval stages into reproductive adulthood. However, if conditions are unfavorable through the second larval stage, worms enter dauer diapause, a state of global and reversible developmental arrest in which precursor cells remain quiescent and preserve developmental potential, anticipating developmental progression if conditions improve. Signaling from neurons, hypodermis, and intestine regulate the appearance and behavior of dauer larvae, and many aspects of developmental arrest of the non-gonadal soma [1, 4, 5]. Here, we show that the decision of somatic gonad blast cells (SGBs) and germline stem cells (GSCs) to be quiescent or progress developmentally is regulated differently from the nongonadal soma: daf-18/PTEN acts non-autonomously within the somatic gonad to maintain developmental quiescence of both SGBs and GSCs. Our analysis suggests that daf-18 acts in somatic gonad cells to produce a “pro-quiescence” signal (or signals) that acts inter se and between the somatic gonad and the germline. The inferred signal does not require DAF-2/Insulin Receptor or maintain quiescence of the nearby Sex Myoblasts, and developmental progression in daf-18(0) does not require Dafachronic Acids. Abrogating quiescence in dauer results in post-dauer sterility. Our results implicate the somatic gonad as an endocrine organ to synchronize somatic gonad and germline development during dauer diapause and recovery, and our finding that PTEN acts non-autonomously to control blast cell quiescence may be relevant to its function as a tumor suppressor in mammals and to combating parasitic nematodes.

Keywords: C. elegans, dauer, diapause, DAF-18, PTEN, gonad, germline, stem cell, quiescence

eTOC

Tenen and Greenwald show that in C. elegans dauer larvae, state of global and reversible developmental arrest, daf-18/PTEN functions cell-nonautonomously in the somatic gonad to regulate a “pro-quiescence” signal(s), which acts within the somatic gonad and between the somatic gonad and the germline to synchronize their development.

Results

daf-18 activity prevents developmental progression of the somatic gonad in dauer larvae

When we set out to investigate if daf-18, the sole C. elegans PTEN gene and a modulator of the insulin signaling pathway [6] is, like daf-16/FoxO, required to maintain quiescence and multipotency of Vulval Precusor Cells of dauer larvae [7], we noticed substantial apparent somatic gonad developmental progression in dauers lacking daf-18 activity that is not seen in dauers lacking daf-16 activity (see below). As maintaining quiescence of the germline also requires daf-18 but not daf-16 [8], the effect on somatic gonad development suggested the possibility that daf-18 may coordinate both aspects of gonadogenesis.

daf-18(0) (null) mutants are defective in dauer formation, but daf-18(0) dauers can be generated using daf-7(e1372)/TGFβ, a parallel input that constitutively induces dauer formation, and dauer larvae may be identified based on their SDS-resistance [9, 10]. In this study, unless otherwise specified, “daf-18(0) dauers” refers to daf-7(e1372); daf-18(ok480null) [11], and “control dauers” are daf-7(e1372); daf-18(+). The arrested gonad in daf-7 control dauers resembles that of genotypically wild-type dauers, and as described below, key findings regarding daf-18(0) dauers were replicated using mutations in daf-2/Insulin receptor and daf-9/steroidogenic hydroxlyase to induce dauer formation.

Gonadogenesis occurs in two phases (Figure 1A). The first phase is completed during the L2 stage with the formation of the somatic gonad primordium, in which the germline stem cells (GSCs) are segregated into two arms, each capped by a distal tip cell (DTC), and a proximal region containing nine somatic gonad blast cells (SGBs) and the Anchor Cell (AC) [12]. In the second phase, beginning in the L3 stage, the SGBs divide and generate the structural cells of the gonad; the AC organizes development of the uterus and vulva; and the DTCs nurture GSC proliferation and lead outward extension of the gonad arms [13]. In dauer larvae, development is suspended between the first and second phases, and both GSCs and SGBs remain quiescent [2].

We marked all somatic gonad cells of daf-18(0) dauers with mCherry-histone (Figure 1 and STAR Methods) and observed a dramatic increase in the number of mCherry-labeled cells, suggesting loss of SGB quiescence (Figure 1B). Expression of cell fate markers for Ventral Uterine precursor cell (VU) and Sheath-Spermatheca precursor cell (SS) descendants characteristic of the second phase of gonadogenesis, including expanded arIs51[cdh-3::gfp] expression indicative of utse (uterine seam syncytium) formation from terminal descendants of VUs, indicated that SGBs undergo normal developmental progression in daf-18(0) dauers (Figure 1C,D). Furthermore, developmental progression of SGBs was not observed prior to daf-18(0) dauer entry, and the penetrance of dauers displaying developmental progression increased with time in dauer, consistent with a role for daf-18 in maintaining SGB quiescence in dauer (Figure S1). The role of daf-18 therefore differs from genes that act in the L2d stage: components of the AMPK pathway, which is required to establish GSC (but not SGB) quiescence [8, 14], and din-1/SHARP, a co-repressor for several transcription factors[15–17], which appears to act cell-autonomously to establish SGB and GSC quiescence in preparation for dauer entry [18].

In promoting dauer formation, daf-18(+) acts via modulation of Insulin/Insulin-like Growth Factor signaling (IIS), leading to increased nuclear localization of DAF-16/FoxO [4]. daf-16 is not required to maintain quiescence of GSCs in dauer [8], but is important for maintaining quiescence of some somatic non-gonadal blast cells in dauer, including the Vulval Precursor Cells [7]. When we tested the requirement of daf-16 for SGB progression, we observed that, whereas 88% of daf-18(0) dauers have expanded arIs51[cdh-3::gfp] utse formation, only 7% of daf-16(mgDf50null); daf-7 do (Figure 1D). This observation suggests that regulation of SGB quiescence in dauer is largely independent of daf-16/FoxO, but it remains possible that a different known [4] or as-yet unknown IIS effector is involved.

daf-18 functions in the somatic gonad primordium to prevent SGB and GSC developmental progression in dauer

The cellular basis for the regulation of dauer entry involves multiple non-gonadal tissues. Environmental conditions detected by sensory neurons regulate the production of insulin-like growth factors, DAF-7/TGFβ, and steroid hormone ligands; these inputs are integrated to control the decision between proceeding to reproductive adulthood or entering dauer diapause [1–3, 5]. Tissue-specific expression of daf-18 activity in neurons, intestine, and the seam cells of the hypodermis, but not muscle, restores the ability to form dauers in daf-18(0) mutants [19].

To ascertain the cellular focus of daf-18 activity for preventing SGB and GSC progression in dauer larvae, we first performed genetic mosaic analysis [20]: random mitotic loss of arEx2399[daf-18(+)], an extrachromosomal array carrying daf-18(+) in a daf-7; daf-18(0) background, created “daf-18(0) mosaic dauers” lacking daf-18(+) activity in defined embryonic founder cells and their descendants (Figure 2 and STAR Methods). SGB progression was assessed using arIs51[cdh-3::gfp], which expands from the AC to the utse near the end of the second phase of gonadogenesis (see Figure 1), and GSC progression was assessed by anatomy (STAR Methods). This analysis (Figure 2A) revealed that progression of both somatic gonad and germline development was blocked in daf-18(0) mosaic dauers that retained the array in the MS lineage, which gives rise to the somatic gonad, even when lineages that generate the germline, intestine, hypodermis, and most of the nervous system lacked the array [and therefore lacked daf-18 activity]. Conversely, daf-18(0) mosaic dauers lacking the array in the MS lineage displayed progression of somatic gonad and germline development even when the lineages forming the germline, intestine, nervous system, and hypodermis retained daf-18(+). These results suggested that daf-18 acts in the somatic gonad to maintain both SGB and GSC quiescence.

Figure 2. — Both control dauers and *daf-18(0)* dauers contain *daf-7(e1372)* and markers.

**(A)** Genetic mosaic analysis. Genetic mosaic dauers lacking *daf-18(+)* activity in defined lineages were isolated from strain GS8095 *daf-7(e1372); daf-18(ok480) arIs51[cdh-3::gfp]; arEx2399[daf-18(+)]* (see STAR Methods for details). *arEx2399* includes fluorescent markers that are visible in E (intestinal) derivatives and body wall muscles. Each circle represents an individual with a simple loss of the array in one lineage; a square represents a compound loss. Red, SGB and GSC progression was observed when the array was lost in lineages leading to the somatic gonad; white, losses in which both SGBs and GSCs remained quiescent, including losses in lineages giving rise to the germline. Note that all eight losses in the MS lineage retained the array in the E lineage, all simple losses in the MS lineage retained the array in all other lineages, and tissue-specific rescue experiments in Figure S2B provide additional evidence against a cellular focus in the intestine or neurons.

SGB progression was scored as expanded *cdh-3::gfp* expression indicative of utse formation (see Figure 1C); GSC progression was scored by DIC. In 23/24 mosaics, the SGBs and GSCs either progressed simultaneously or maintained quiescence.

**(B)** Tissue-specific expression of *daf-18(+)* in the somatic gonad or DTCs rescues SGB and GSC quiescence in dauer. SGB progression was scored by expanded *aris51[cdh-3::gfp]* expression indicative of utse formation, and GSC progression was scored by DIC (see Figure 1C and STAR Methods). A representative transgene expressing in all somatic gonad cells (*arTi195, ckb-3p::daf-18(+)::T2A::tagBFP2*; purple) or DTCs only (*arTi118, hlh-12p::daf-18(+)::T2A::tagBFP2*; red) is shown; data for additional, independent transgenes with these constructs are shown in Figure S2A. (***) P ≤ 0.001 by two-tailed Fisher’s exact test.

**(C)** Photomicrograph showing DTC-specific expression of *daf-18(+)* rescues SGB and GSC progression in *daf-18(0)* dauers. This individual is of genotype *daf-18(0); daf-7; arEx2416[hlh-12p::S::daf-18::T2A::tagBFP2::unc-54 3’UTR]; arIs51[cdh-3p::gfp]*, with tagBFP2 (produced from a bicistronic message due to the T2A linker [51]) marking the DTCs (see STAR Methods). Gonad development has not progressed, as is evident from the presence of an AC expressing *arIs51[cdh-3::gfp]* in the proximal gonad rather than expanded expression associated with a utse, and the small size of the gonad arms (compare with Figure 1).

See also Figure S2.

We tested this inference by restoring DAF-18 to the somatic gonad primordium using the ckb-3 promoter [21]. In dauer larvae, ckb-3p::mCherry-histone is seen in all cells of the somatic gonad primordium and in only three other cells, tentatively identified as MS-derived pharyngeal muscle segment pm6 of the pharynx (STAR Methods). Three independent extrachromosomal arrays and a single-copy insertion transgene of ckb-3p::daf-18(+) restored SGB and GSC quiescence to daf-18(0) dauers (Figure 2B; Figure S2A), consistent with daf-18(+) function in the somatic gonad preventing both somatic gonad and germline developmental progression. Additional tissue-specific rescue experiments supported the conclusion that the presence of daf-18 activity in the intestine or nervous system is not sufficient to maintain SGB and GSC quiescence (Figure S2B).

daf-18 has a diffuse cellular focus within the somatic gonad

The above analysis indicated that daf-18 acts in the somatic gonad to maintain GSC quiescence non-autonomously. Here, we ask if there is a specific cellular focus within the somatic gonad for maintaining GSC and SGB quiescence.

In each arm, the terminally-differentiated DTC forms a niche for the GSCs, and in the L4 stage of continuous development, responds to nutritional signals to regulate the number of germ cells [22]. Therefore, it seemed plausible that daf-18 might act in dauer DTCs to regulate GSC quiescence non-autonomously, but act autonomously within the SGBs to regulate their quiescence. Remarkably, expression of DAF-18 specifically in DTCs of daf-18(0) dauers restored both GSC and SGB quiescence (Figure 2B,C). Thus, daf-18 activity in the DTCs is sufficient to maintain quiescence non-autonomously of both SGBs and GSCs. However, when we created a “floxed” allele, daf-18 [loxP>daf-18(+)>loxP], and generated “excision mosaics” by expressing Cre recombinase specifically in the DTCs [23–25] (Figure 3A), both SGBs and GSCs remained quiescent, suggesting that the DTCs are not the sole cellular focus in the somatic gonad for maintaining blast cell quiescence (Figure 3B).

Figure 3. — All dauers contain *daf-7(e1372); daf-18(ok480)*, markers, excisable *daf-18(+)*, and Cre drivers.

**(A)** Strategy to generate excision mosaics. The conditional allele *arTi179, daf-18 [loxP>daf-18>loxP]*, restores normal dauer development in a *daf-18(0); daf-7* background. Tissue-specific expression of Cre recombinase using the single-copy insertion drivers shown (box) generates excision mosaics in which *daf-18(+)* is removed from the DTCs [*hlh-12*p], all somatic gonad cells [*ckb*-3p], or variably in all/most or no somatic gonad cells [*hlh-1*p] because of transient expression in MS granddaughters [52, 53]. None of the Cre drivers used caused excision in the germline. For more details of excision patterns observed with these drivers, see STAR Methods. *daf-18(0)* excision mosaics were obtained using a cross strategy (box) that (i) eliminated potential maternal contribution of *daf-18(+)* by providing the DAF-18-expressing *loxP>daf-18(+)>loxP* floxed locus through the male and (ii) required a single excision event involving the *daf-18 [loxP>daf-18>loxP]* locus present in F1 heterozygous progeny, so that all GFP-positive DTCs lacked *daf-18(+)* activity. For further details about generation of transgenes and additional observations about the full effects of the drivers used see STAR Methods.

**(B)** SGB and GSC progression in excision mosaics. The excision pattern induced by each driver shown was confirmed by both GFP expression from the excision event involving the single *daf-18 [loxP>daf-18>loxP]* locus (see above) in the mosaics scored and analysis of *heIs105*, an established recombination reporter [25] (STAR Methods). SGB, somatic gonad blast cell; DTC, distal tip cell; GSC, germline stem cell. SGB division progression was inferred by substantially increased number of cells expressing *ckb-3p::mCherry::H2B* and concomitant expansion by DIC, and germline divisions were scored by DIC (see Figure 1 B,C). (***) P ≤ 0.001 by two-tailed Fisher’s exact test.

See also Figure S3.

Genetic ablation of the DTCs (along with the AC) using hlh-2(RNAi) [26, 27] in daf-18(+) and daf-18(0) backgrounds supports this conclusion. The absence of DTCs did not abrogate quiescence of the SGBs in daf-18(+) dauers (Figure S3A) and did not restore quiescence to the SGBs in daf-18(0) dauers (Figure S3B). GSC quiescence or progression could not be evaluated, as DTCs are required for continued GSC divisions [13]. These observations also suggest that the AC, the only other terminally differentiated cell present in the somatic gonad primordium, is not the sole redundant cellular focus along with the DTCs.

Since daf-18 expression in the somatic gonad or in the DTCs is sufficient to rescue daf-18(0), but daf-18 activity in the DTCs is not necessary, we conclude that there is a diffuse cellular focus within the somatic gonad. Consistent with this view, excision mosaics lacking daf-18 activity in all somatic gonad cells caused both SGBs and GSCs to lose quiescence (Figure 3). In addition, the excision mosaics expressed GFP-H2B under the control of the daf-18 promoter, indicating that daf-18 normally is expressed in all somatic gonad cells in dauer. Finally, as the transgene fully rescues daf-18(0), and the somatic gonad excision mosaics that display progression retained the intact loxP>daf-18(+)>loxP transgene in the germline (STAR Methods), our observations further support a somatic gonad focus for daf-18 rather than in the germline in maintaining GSC quiescence in dauer.

Loss of daf-18 permits SGB and GSC progression in other dauer-constitutive mutant backgrounds

To test if the effects described above depend on the daf-7 dauer-constitutive mutant background, we also tested the effect of daf-18(0) in dauer-constitutive mutant backgrounds that compromise one of the other major signaling pathways that regulate dauer entry, Insulin/Insulin-like Growth Factor signaling (IIS) or steroid hormone receptor signaling [1, 4]. The analysis indicates that the requirement for daf-18 to maintain SGB and GSC quiescence in dauer is not a special property of the daf-7 background, and each also has additional implications.

In the presence of food, agonistic insulin-like peptides activate the sole Insulin Receptor (InsR) ortholog DAF-2 to promote continuous development [2, 4]. In C. elegans, there are about 40 genes encoding insulin-like peptides, some agonistic and others antagonistic [28–30]. Thus, a potential mechanism by which daf-18 may promote quiescence would be to promote the expression or activity of an antagonistic insulin in the somatic gonad primordium. This role, or any other role for IIS in this process, could be revealed if loss of daf-2 prevented developmental progression in daf-18(0). We used the reference allele daf-2(e1370ts) to drive dauer formation at the restrictive temperature, and as daf-18(0) suppresses dauer formation of daf-2(e1370ts), we created genetic mosaics for daf-18(0) by loss of arEx2399[daf-18(+)]. In mosaics retaining arEx2399[daf-18(+)] in the MS lineage, both SGBs and GSCs remained quiescent; in mosaics lacking arEx2399[daf-18(+)] in the MS lineage, both SGBs and GSCs developmentally progressed (Figure 4A; Figure S4). This result supports the conclusion from our analysis of daf-7; daf-18(0) dauers that daf-18 acts in the somatic gonad to maintain SGB and GSC quiescence, and that the inferred pro-quiescence somatic gonad endocrine signal regulated by daf-18 is not likely to be an inhibitory insulin-like peptide.

Figure 4. — **(A)** Effect of *daf-18(0)* in dauers formed by *daf-c* mutations affecting IIS or NHR signaling. SGB progression was inferred in all dauer larvae based on expanded *arIs51[cdh-3::gfp]* expression indicative of utse formation and overall anatomy. GSC progression was inferred based on overtly increased size and GSC number in the gonad arms (STAR Methods).

To evaluate IIS, we tested whether SGBs and GSCs progress in *daf-18(0)* dauers with the canonical loss of function mutation in *daf-2*/IR, *daf-2(e1370)*. Since *daf-18(0)* suppresses constitutive dauer formation caused by *daf-2(e1370)*, we created *daf-18(0)* mosaic dauers lacking *daf-18(+)* in the MS lineage. SGB and concomitant GSC progression was observed in *daf-18(0)* dauers lacking DAF-18(+) in the MS lineage, and was not observed in dauers retaining DAF-18(+) in the MS lineage. *daf-7(e1372)* and *daf-2(e1370)* are temperature-sensitive mutants that form dauers constitutively at 25°C.

The null allele *daf-9(dh6)* was used to prevent the production of DA ligands for DAF-12/NHR, keeping it in the unliganded state the promotes dauer development. Because *daf-9(dh6)* forms dauers constitutively, strains were maintained using a rescuing extrachromosomal array, which is lost meiotically to create homozygous *daf-9(0)* non-mosaic dauers [54] (STAR Methods). We note also that, in *daf-18(0); daf-9(0)* dauers, significantly more GSC progression was observed than SGB progression (P ≤ 0.01).

(***) P ≤ 0.001, (**) P ≤ 0.01 by two-tailed Fisher’s exact test.

**(B)** Tissue-specific rescue of somatic gonad quiescence does not restore SM quiescence to *daf-18(0)* dauers. Top, *daf-7(e1372)* “control dauer” with a quiescent gonad, showing *cdh-3::gfp* expression in the AC and the two SMs, labeled with *hlh-8p::mCherry*. Bottom, “Rescued gonad” in which *ckb-3p::daf-18(+)* restores quiescence to the somatic gonad and GSCs (see also Figure 2B) without restoring quiescence to SMs, as evidenced by multiple SM descendants labeled with *hlh-8p::mCherry*. For detailed genotypes see Methods. (***) P ≤ 0.001 by two-tailed Fisher’s exact test compared with *daf-7(e1372)*.

**(C)** Model for *daf-18* function in regulating quiescence of GSCs and SGBs in dauer. Our analysis indicates that loss of *daf-18* in the somatic cells of the gonad leads to concomitant loss of both SGB quiescence and GSC quiescence. We therefore infer that *daf-18(+)* activity in somatic gonad cells regulates the activity or production of a signal or signals to the germline and *inter se* to promote quiescence. We do not know if there is a single pro-quiescence signal that acts on both tissues or if there are distinct signals. Since restoration of *daf-18(+)* specifically to the somatic gonad rescues both somatic gonad and germline quiescence, a simple model is that there is a single signal that coordinates developmental progression of both tissues.

See also Figure S4.

Steroids called Dafachronic Acids (DAs) are essential ligands for the nuclear hormone receptor DAF-12, which promotes continuous development in the presence of DA and dauer development in the absence of DA [1]. The production of DAs requires the steroidogenic hydroxylase DAF-9, which acts downstream of both IIS and DAF-7/TGFβ signaling in regulating the dauer entry decision (Figure 4A) [15, 31, 32]. daf-9(0) mutants constitutively become dauer larvae, and daf-18(0); daf-9(0) dauers showed significantly increased SGB and GSC progression compared to daf-9(0) dauers (Figure 4A), indicating that developmental progression in the absence of DAF-18 does not absolutely depend on DAs. Curiously, we observed that a significant number of daf-18(0); daf-9(0) dauers displayed GSC progression without concomitant SGB progression (Figure 4A), an uncoupling of these processes we have not observed in other daf-18(0) dauer larvae.

The somatic gonad does not regulate sex myoblast quiescence in dauer larvae

Like the SGBs of the somatic gonad, the sex myoblasts (SMs) are mesodermal in origin. The SMs are born in the L1 stage and remain quiescent in the L2 stage, during which time they migrate so as to align with the center of the proximal gonad, and re-enter the cell cycle in the L3 stage [33]. This migration is guided by an FGF-like signal from the somatic gonad [34]. We marked SMs using arTi133[hlh-8p::gfp] (STAR Methods) and observed that in daf-7 control dauers, the SMs remained quiescent in their normal positions adjacent to the proximal gonad; in contrast, in daf-18(0) dauers, the SMs generated multiple descendants, indicating that they lost quiescence (Figure 4B and STAR Methods). To test if the daf-18-regulated endocrine signal produced by somatic gonadal cells also regulates SM quiescence, we restored quiescence to the SGBs and GSCs by expressing daf-18(+) in the somatic gonad cells in a daf-18(0) mutant background. We observed numerous marked SM descendants, indicating that SM quiescence was not restored (Figure 4B), and suggesting the possibility that the pro-quiescence endocrine signal produced in the somatic gonad primordium may not be freely secreted or is unable to cross the basement membrane barrier that surrounds the entire gonad.

Post-dauer defects in daf-18 somatic gonad excision mosaics reveal the importance of maintaining developmental quiescence in dauer

To determine the impact of inappropriate developmental progression on post-dauer reproductive system development, we examined individual excision mosaic dauers in which a Cre driver induced excision of loxP>daf-18(+)>loxP in somatic gonad cells and in control dauers (no Cre driver) after SDS-selection and recovery at 15°C (STAR Methods). In recovered control dauers, 16/16 post-dauer adults were fertile and 15/16 laid eggs normally. In contrast,12/14 excision mosaics lacking daf-18 in the somatic gonad were overtly abnormal: 9/14 were sterile (***p < 0.001 vs control), and 3/14 were fertile but egg-laying defective. Thus, the loss of developmental quiescence due to lack of daf-18 activity in the somatic gonad of dauer larvae has adverse consequences for post-dauer fertility, underscoring the importance of the as-yet unknown endocrine signal in dauer life history.

Discussion

Our finding that daf-18/PTEN acts cell non-autonomously within the somatic gonad primordium to regulate the decision of both SGBs and GSCs to be quiescent or to resume development implicates the somatic gonad as an endocrine organ in dauer larvae, with somatic gonadal cells signaling both inter se and to the germline to regulate developmental progression. In principle, daf-18 activity could normally be required for somatic gonad cells to produce a “pro-quiescence” signal, or loss of daf-18 could lead to ectopic production of a “pro-progression” signal. Because expression of daf-18(+) in DTCs is sufficient to restore quiescence to the SGBs and GSCs even while the remainder of the somatic gonad and other tissues are daf-18(0), we favor the interpretation that daf-18 activity in dauer normally promotes the production or activity of a single pro-quiescence signal, or discrete ones to the SGBs vs. GSCs, from somatic gonad cells (Figure 4C).

In many situations, PTEN acts autonomously, e.g. to regulate cell size in the Drosophila ommatidium [35] and mammalian stem cell quiescence [e.g. [35–39]]. Curiously, in mammals, one PTEN isoform has a poly-arginine stretch that mediates its secretion [40] and PTEN has been observed in extracellular vesicles [41]; although DAF-18 does not have any poly-arginine regions, extracellular vesicles have been observed in C. elegans [reviewed in [42]] raising the possibility that DAF-18 movement per se could explain the apparent non-autonomy we observed. However, even if there is any such movement of DAF-18, it must be highly constrained or specifically directed, because retaining daf-18(+) in the germline while the somatic gonad is daf-18(0) does not rescue either GSC or SGB quiescence, and expressing daf-18(+) in the somatic gonad restores quiescence to SGBs and GSCs but not the nearby SMs.

Indeed, our analysis indicates that any signal regulated by daf-18 is specific to gonadal cells and/or has a limited range, and in the case of the communication between somatic gonad and GSCs, directionality. Regardless of mechanism, it is apparent that coordinated development of soma and germline mediated by daf-18 is important for post-dauer fecundity. We envisage that the somatic gonad cells receive the inputs from non-gonadal soma that regulate the global dauer entry/exit decision, such that the gonadal endocrine activity/activities diagrammed in Figure 4C not only ensure that development of somatic gonad and germline are coordinated with each other, but also with global developmental progression. One interesting question for the future is the relationship between the pro-quiescence signal we infer from our studies and the AMPK pathway [8, 14] in establishing GSC quiescence in dauer larvae.

The dauer larva is a highly specialized form for survival under adverse conditions, and represents an alternative life history that reflects the integration of information about population density and temperature as well as availability of food [2]. C. elegans development is also simply suspended at other points in response to food deprivation [43–45]. The best studied paradigm for developmental suspension induced by food deprivation is L1 arrest. Like entry into dauer diapause, L1 arrest requires the activity of daf-16/FoxO and daf-18/PTEN, and similar to the dauer germline, GSC quiescence in L1 arrest requires the activity of daf-18 but not daf-16 [43]. Tissue-specific expression of daf-18(+) in the hypodermis is sufficient to restore quiescence of non-gonadal somatic precursors, but not of the somatic gonad precursors Z1 and Z4 or of the germline precursors [46]. It will be interesting to test if restoration of daf-18 to Z1 and Z4 also restores quiescence of both somatic and germline precursors during L1 arrest, and eventually, to determine if the same signals are regulated by daf-18 in both the dauer and L1 arrest paradigm.

Quiescence is a fundamental property of stem cell biology, aging and cancer [47], and PTEN is an important tumor suppressor that is commonly mutated in human cancer [48]. Our finding that PTEN acts non-autonomously in dauer larvae to control the quiescence of blast cells, potentially by regulating the production of a “pro-quiescence” signal, may be relevant to its function as a tumor suppressor in some human tissue contexts. Furthermore, the dauer larva is related to the infectious stage of many parasitic nematodes, suggesting that inhibition of DAF-18/PTEN function may be an approach to diminishing parasitic nematode infection [49, 50].

STAR Methods

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Iva Greenwald (isg4@columbia.edu).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

C. elegans strains and transgenes

See Key Resources Table for the full list of strains. Strains carrying temperature-sensitive dauer-constitutive mutations were maintained at 15-20°C and shifted to 25°C as described in the Method Details. The dauer-defective mutations daf-16(mgDf50) I [daf-16(0)] and daf-18(ok480) IV [daf-18(0)] and the dauer-constitutive mutations daf-7(e1372) III, daf-2(e1370) III, daf-9(dh6) X [daf-9(0)] are described in WormBase.

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Experimental Models: Organisms/Strains
N2: wild type	Caenorhabditis Genetics Center (CGC)	WB Strain: N2
GS8052: daf-7(e1372) III; arIs51[cdh-3::gfp] IV	This paper	N/A
GS8925: daf-7(e1372) III; daf-18(ok480) arIs51[cdh-3::gfp] IV	This paper	N/A
GS8024: daf-7(e1372) III; daf-18(ok480) arIs51[cdh-3::gfp] IV	This paper	N/A
GS8292: arTi112[ckb-3p::mCherry::his-58::unc-54 3'UTR]	This paper	N/A
GS8440: daf-7(e1372) III; arTi112[ckb-3p:mCherry::H2B] V	This paper	N/A
GS8441: daf-7(e1372) III; daf-18(ok480) IV; arTi112[ckb-3p::mCherry::H2B] V	This paper	N/A
GS7917: daf-7(e1372) III; kuIs29[egl-13::gfp] V	This paper	N/A
GS7918: daf-7(e1372) syIs80[lin-11::gfp] III	This paper	N/A
GS8047: daf-7(e1372) III; daf-18(ok480) IV; kuIs29[egl-13::gfp] V	This paper	N/A
GS8048: daf-7(e1372) syIs80[lin-11::gfp] III; daf-18(ok480) IV	This paper	N/A
GS8085: daf-7(e1372) III; tnIs6[lim-7::GFP + rol-6(su1006)]	This paper	N/A
GS8635: daf-7(e1372) III; daf-18(ok480) IV; tnIs6[lim-7::GFP + rol-6(su1006)]	This paper	N/A
GS8593: arIs51[cdh-3::gfp] IV; daf-9(dh6) X; dhEx24[daf-9(+), sur-5::GFP]	This paper	N/A
GS8594: daf-18(ok480) arIs51[cdh-3::gfp] IV; daf-9(dh6) X; dhEx24[daf-9(+), sur-5::GFP]	This paper	N/A
GS8095: daf-7(e1372) III; daf-18(ok480) arIs51 IV; arEx2399	This paper	N/A
GS8092: arEx2399[daf-18(+)]	This paper	N/A
PS3808: unc-119(ed4) syIs80[u-119(+)pPGF11.13(lin-11::gfp)]III	CGC	WB Strain: PS3808, RRID:WB-STRAIN:PS3808
MH1317: kuIs29[unc-119(+);egl-13::gfp]	CGC	WB Strain: MH1317; RRID:WB-STRAIN:MH1317
DG1575: tnIs6[lim-7::GFP + rol-6(su1006)]	CGC	WB Strain: DG1575
GS8458: arTi133[hlh-8p::ERK(3)KTR-Clover-T2A-mCherry-his-11]	[53]	N/A
GS8939: arTi133[hlh-8p::ERK(3)KTR-Clover-T2A-mCherry-his-11] II; daf-7(e1372) III	This paper	N/A
GS8940: arTi133[hlh-8p::ERK(3)KTR-Clover-T2A-mCherry-his-11] II; daf-7(e1372) III; daf-18(ok480) IV	This paper	N/A
GS9082: arTi133 II; daf-7(e1372) III; arIs51 IV	This paper	N/A
GS9083: arTi133 II; daf-7(e1372) III; daf-18(ok480) arIs51 IV; arTi195 V	This paper	N/A
GS8525: daf-7(e1372) arIs131[lag-2p::2xnls::yfp] III; arTi112[ckb-3p::mCherry::H2B] V; nre-1(hd20) lin-15b(hd126) X	This paper	N/A
GS8604: daf-7(e1372) arIs131[lag-2p::2xnls::yfp] III; daf-18(ok480) IV; arTi112[ckb-3p::mCherry::H2B] V; nre-1(hd20) lin-15b(hd126) X	This paper	N/A
GS8421: arTi118[hlh-12p::daf-18::T2A::tagBFP2::unc-54 3'UTR] II	This paper	N/A
GS8422: arTi118[hlh-12p::daf-18::T2A::tagBFP2::unc-54 3'UTR] II; daf-7(e1372); daf-18(ok480) arIs51	This paper	N/A
GS8642: daf-7(e1372) III; daf-18(ok480) arIs51 IV; arTi195 V	This paper	N/A
GS8200: daf-7(e1372); daf-18(ok480) arIs51; arEx2413[ckb-3p::daf-18cDNA::T2A::tagBFP2::unc-54 3'UTR]	This paper	N/A
GS8201: daf-7(e1372); daf-18(ok480) arIs51; arEx2414[ckb-3p::daf-18cDNA::T2A::tagBFP2::unc-54 3'UTR]	This paper	N/A
GS8202: daf-7(e1372); daf-18(ok480) arIs51; arEx2415[ckb-3p::daf-18cDNA::T2A::tagBFP2::unc-54 3'UTR]	This paper	N/A
GS8203: daf-7(e1372); daf-18(ok480) arIs51; arEx2416[hlh-12p::daf-18cDNA::T2A::tagBFP2::unc-54 3'UTR]	This paper	N/A
RG1078: daf-18(ok480) IV; veEx344 [pMF248(Prgef-1::daf-18 cDNA::myc::daf-18 3'UTR) (3 ng/μl) + str-1::gfp (100 ng/μl)]	[67] and CGC	N/A
RG1083: daf-18(ok480) IV; veEx349 [pMF253(Pges-1::daf-18 cDNA::myc::daf-18 3'UTR) (16 ng/μl) + str-1::gfp (100 ng/μl)]	[67] and CGC	N/A
YB409: daf-18(ok480) IV; tdEx239 [pMF273(Pdaf-18::daf-18::myc::daf-18 3'UTR) (15 ng/μl) + str-1::gfp (90 ng/μl)]	[67] and CGC	N/A
GS8439: daf-7(e1372) III; daf-18(ok480) arIs51 IV; veEx344[Prgef-1::daf-18 cDNA::myc::daf-18 3'UTR]	This paper	N/A
GS8423: daf-7(e1372); daf-18(ok480) arIs51; veEx349[Pges-1::daf-18 cDNA::myc::daf-18 3'UTR]	This paper	N/A
GS8643: daf-7(e1372); daf-18(ok480) arIs51 IV; tdEx239 [Pdaf-18::daf-18::myc::daf-18 3'UTR]	This paper	N/A
GS8588: arTi179 (may be heterozygous)	This paper	N/A
GS8730: arTi179 I; daf-7(e1372) III; daf-18(ok480) IV; arTi112 V	This paper	N/A
GS8577: arTi168 (may be heterozygous)	This paper	N/A
GS8793: arTi235[pCT42(hlh-1(3130)p::Cre(opti)::tbb-2 3'UTR)] (may be heterozygous)	This paper	N/A
GS8794: arTi236[pCT43(ckb-3p::Cre(opti)::tbb-2 3'UTR)] (may be heterozygous)	This paper	N/A
GS8850: daf-7(e1372) III; daf-18(ok480); arTi168 X	This paper	N/A
GS8789: daf-7(e1372) III; daf-18(ok480) IV; arTi235[pCT42(hlh-1(3130)p::Cre(opti)::tbb-2 3'UTR)]	This paper	N/A
GS8799: daf-7(e1372) III; daf-18(ok480) IV; arTi236[pCT43(ckb-3p::Cre(opti)::tbb-2 3'UTR)]	This paper	N/A
GS8685: heIs105 IV (5x bx from SV1361)	This paper	N/A
GS8851: heIs105; arTi168 X	This paper	N/A
GS8796: heIs105 IV; arTi235[pCT42(hlh-1(3130)p::Cre(opti)::tbb-2 3'UTR)]	This paper	N/A
GS8797: heIs105 IV; arTi236[pCT43(ckb-3p::Cre(opti)::tbb-2 3'UTR)]	This paper	N/A
GS9117 : daf-2(e1370); daf-18(ok480) arIs51[cdh-3::gfp]; arEx2399[daf-18(+)]	This paper	N/A
SV1361: unc-119(ed3) III; heIs105 IV	CGC	WB Strain: SV1361; RRID:WB-STRAIN:SV1361
CB1372: daf-7(e1372) III	CGC	WB Strain: CB1372; RRID:WB-STRAIN:CB1372
GS5998: daf-18(ok480)	This paper	N/A
Chemicals, Peptides, and Recombinant Proteins
BD BactoAgar	Fisher	DF0140-07-4
Calcium Chloride, Anhydrous	VWR	97062-59
Cholesterol	MP	101382
Magnesium Sulfate, Anhydrous	Avantor-Fisher	2506-01
Potassium Phosphate, Dibasic	Avantor-Fisher	4012-05
Tetramisole hydrocholoride (levamisole)	Sigma-Aldrich	L9756-10G

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The following transgenes were used to mark cells of the somatic gonad primordium and to ascertain their developmental arrest or progression:

arTi112[ckb-3p::mCherry-H2B] is a single-copy insertion transgene generated with the assistance of Justin Shaffer that marks all cells of the somatic gonad primordium. As in all other constructs with the ckb-3 promoter, a synthetic intron “S” was included to increase expression efficiency [see below; [55, 56]] The ckb-3p [21] drives highest expression in Z1 and Z4 and becomes progressively weaker as the lineages progress.

arIs51[cdh-3p::gfp] [26] is expressed in the AC in the L2 and L3 stages of continuous development and in wild-type starved, daf-7(e1372), daf-2(e1370) and daf-9(dh6) dauers. GFP expands to the utse in the early L4 stage of continuous development [57] and in daf-18(0) dauers as indicated.

syIs80[lin-11::gfp] [58] and kuIs29[egl-13::gfp] [59] mark π cells and their daughters.
tnIs6[lim-7::gfp] marks the descendants of the sheath-spermathecal precursor (SS) [60].

arTi133[hlh-8p::ERK-KTRmClover::T2A::mCherry::H2B], designated arTi133[hlh-8p::mCherry::H2B] in the text, is expressed in the SMs and their descendants [61], and is expressed in the two SMs in dauer.

Additional transgenes generated during the course of this study for mosaic analysis and tissue-specific rescue experiments are described in the Method Details below.

METHOD DETAILS

Identifying and scoring dauer larvae

Unless otherwise specified, dauer larvae carrying dauer-constitutive (daf-c) mutations [daf-7, daf-2, or daf-9] were obtained by treating gravid hermaphrodites with bleach to release eggs using a standard protocol [62]. The eggs were incubated at 25° C on standard NGM plates containing E. coli [63]. When scored approximately 72 hours later, dauers have been arrested for at least 24 hours; these are designated “1-day dauers.” When scored approximately 96 hours after eggs are shifted to 25° C, dauers with daf-c alleles have been in dauer for at least 48 hours; these are designated 2-day dauers. When scoring double mutant dauers carrying daf-c and dauer-defective [daf-d] mutations [daf-18(0) or daf-16(0)], the daf-c controls were set up and scored in parallel to daf-c; daf-d strains.

Dauer larvae induced using daf-c mutations were identified by SDS selection: incubation in 1% SDS for at least 10 minutes kills all non-dauer stages except for eggs, from which dauer larvae can easily be distinguished [64, 65]. Dauers induced by starvation were identified based on dauer morphology: pharyngeal constriction, radial constriction, and the presence of dauer alae.

Imaging

For all scoring and imaging, larvae were mounted on agarose pads and immobilized in 10 mM levamisole. To characterize arTI112[ckb-3p::mCherry::H2B] in daf-18(0) dauers (Figure 1B, 1D), mCherry-expressing cells were counted and imaged by collecting Z-stacks of mCherry fluorescence with a Zeiss spinning disk confocal dual camera system. For all other experiments, larvae were scored and/or imaged with a 40× Plan-Neo or a 63× Plan-Apo objective, either on a Zeiss Axio Imager Z1 microscope with a Hamamatsu Orca-ER camera or a Zeiss Axio Imager D1 microscope with an AxioCam MRm. An X-Cite 120Q light source from EXFO photonics solutions was used for illumination.

We note that for strains in which both tagBFP2 and GFP were assessed, for illuminating tagBFP2, we used a filter that did not admit GFP from transgene markers (excitation 379-401nm, beamsplitter 420nm, emission 435-485nm) and for illuminating GFP, we used a filter that did not admit tagBFP2 (excitation 450-490nm, beamsplitter 495nm, emission 500-550nm).

Evaluating SGB progression using fluorescent markers

arIs51[cdh-3::gfp]: characterized in daf-18(0) dauers (Figure 1) and used to assess SGB progression in all mosaic analysis (Figures 2, 4 and Supplemental Figure 4), tissue-specific rescue (Figure 3, Supplemental Figure 3), alternative daf-c backgrounds (Figure 4 and Supplemental Figure 4), and L2d/L2d-Dauer molt/Dauer timing experiments (Supplemental Figure 1).

In continuous development, arIs51[cdh-3::gfp] is expressed in the AC of the somatic primordium in the L2 stage, and remains restricted to the AC until it expands to multiple cells of the utse in the L4 stage. Dauer SGBs were considered to have progressed if they had expanded expression of GFP in multiple cells confirmed by DIC microscopy. Details on scoring arIs51[cdh-3::gfp] in L2d/L2d-Dauer molt/Dauer timing experiments are described in Figure S1.

Control dauers for daf-18 experiments are from strain GS8052 daf-7(e1372); arIs51[cdh-3::gfp] unless otherwise specified. daf-18(0) dauers are from strain GS8925 daf-7(e1372); daf-18(ok480) arIs51[cdh-3::gfp] unless otherwise specified.

arTi112[ckb-3p::mCherry::H2B]: normally, mCherry-H2B is evident in the 12 cells of the wild-type or daf-c mutant dauer somatic gonad primordium; dauers with expression in ≥20 cells were considered to have had SGB division, indicative of developmental progression.

syIs80[lin-11::gfp] and kuIs29[egl-13::gfp]: These markers are not expressed in the somatic primordium in wild-type or daf-c dauers (see above); any marker expression in multiple cells of the somatic gonad indicates SGB progression.

tnIs6[lim-7::gfp]: This marker is expressed broadly in sheath cells and their extensions that surround the germ cell nuclei (see above), so Sheath-Spermathecal (SS) blast cell progression was inferred from any expanded expression consistent with sheath cell morphology.

Scoring GSC quiescence and progression in daf-7(e1372) or daf-2(e1370) dauers

GSC quiescence and progression had been extensively characterized in daf-7(e1372) and daf-2(e1370) control dauers, as well as daf-18(0) or daf-18 partial loss of function mutant dauers [8]. In these studies, precise counts of the number of GSCs revealed that the increase in numbers were accompanied by a corresponding overt increase in germline size by anatomy, indicating that anatomical enlargement of the gonad arms was a valid metric for GSC progression.

In control dauers, we observed that gonad arms arrested with a consistent width, length, extension, and overall size, containing approximately 30 GSCs total as previously reported (Figure 1B,C; [8]). In contrast, in most daf-18(0) dauers, one or both gonad arms appeared substantially enlarged, displaying increased width and a large number of GSCs as assessed by microscopy. GSC progression was frequently indicated by dramatic extension of the germline arms that could reach the pharynx or tail of the dauers; such extension was also accompanied by increased GSC numbers in daf-18(0) dauers as previously reported [8].

Gonad size was scored categorically as no/mild increase (no progression, approximately ≤50 μm extension and ≤11 μm width when lateral), or as having an overt increase (approximately ≥ 80 μm extension or ≥14 μm width when lateral) compared to controls. We conservatively considered dauers with a mild increase as not having GSC progression to guard against any potential distortion due to daf-18(0) defects in radial constriction. In dauers with moderate or extreme enlargement, evident by increased width or extension to an extent never observed in control dauers, GSCs were considered to have progressed. See Figure 1 for examples of dauers with moderate (Figure 1C) and extreme (Figure 1B) increases in germline width and length.

Scoring GSC quiescence and progression in daf-9(dh6) dauers

The daf-9 null allele, daf-9(dh6), prevents DA production and results in constitutive dauer formation [31]. Strains containing daf-9(0) were maintained with a rescuing extrachromosomal array, and meiotic loss of the array in the parent germline led to segregation of homozygous daf-9(0) dauers [54].

daf-18(0); daf-9(0) dauers were scored categorically as no/mild increase (extension of 70-80 μm, no progression), or as having an overt increase (extension over 90 μm) compared to daf-9(0) control dauers. We note that daf-9(0) dauers arrest with more GSCs than daf-7(e1372) or daf-2(e1370) dauers; however, there is no discernable difference in germline size, extension, or width between early dauer arrest (<12 hours in dauer, n=11, scored approximately 48 hours after eggs were shifted to 25°) and 1-day dauers (n=20, scored 72 hours after the shift), indicating that there is no further progression of germline development in the daf-9(0) control dauers.

daf-18 mosaic analysis for cellular focus

Strain GS8095 [daf-7(e1372); daf-18(ok480) arls51[cdh-3::gfp]; arEx2399[daf-18(+)] was used for genetic mosaic analysis shown in Figure 2A. Non-mosaic individuals were fully rescued for daf-18 defects, i.e. they formed dauer larvae that displayed radial body constriction, pharyngeal constriction, and somatic gonad and germline quiescence (n=10).

Generation of arEx2399[daf-18(+)] for mosaic analysis

arEx2399 is a simple extrachromosomal array. daf-18(+) was provided by a PCR product amplified from N2 genomic DNA, including the entirety of the 5’ UTR and 3’ UTR up to the coding regions of the genes immediately upstream and downstream of daf-18 (IV:419,952 to 426,169, a total of 6218 base pairs; Wormbase release number WS267). The PCR product (25 ng/μl) was coinjected with pDS266(g/o-1p::tagRFP, 25 ng/μl, kindly provided by Dr. Daniel Shaye), pMS102 [lag-2p(FL)::2xnls::tagRFP, 25 ng/μl; [66]], and p716(myo-3p::mCherry, 25 ng/μl) [67] into the germline of N2 hermaphrodites. The injected hermaphrodites were placed at 15°C, and 7 days later, individual descendants were isolated from each injected parent and examined for potential array-carrying progeny based on expression of the myo-3p:: mCherry and glo-1p::tagRFP coinjection markers. The lag-2p(FL)::2xnls::tagRFP could not be visualized in the expected tissues (neurons and somatic gonad), likely due to the greater relative brightness of myo-3p::mCherry. The array arEx2399 was selected to generate strain GS8095.

Generation of daf-18(0) mosaic dauers

To generate mosaics, we relied on random mitotic loss of arEx2399, an extrachromosomal array carrying daf-18(+) in strain GS8095. Such “daf-18(0) mosaic dauers” lack daf-18(+) activity in different embryonic founder cells and their descendants, with the losses inferred by fluorescent markers also carried on arEx2399[daf-18(+)]: myo-3p::mCherry, expressed in all body wall muscles [68] (with defined muscle cells descending from the founder cells ABp, MS, C and D) and glo-1p::tagRFP, expressed in the intestine [69], which is derived solely from E [70]. The germline derives solely from the founder cell P₄; an array loss in P₂ would mean absence of the array in the germline precursor P₄, and a P₂ loss was inferred based on absence of the array in C- and D-derived body wall muscle cells (see Figure 2). Such P₂ losses give confidence that the lack of daf-18 activity does not abrogate GSC quiescence. Conversely, retention of the array in C and D implies likely retention in P₄ and therefore that the arEx2399[daf-18(+)] array was present in the GSCs.

Mosaic dauers were obtained by allowing 40 GS8095 daf-7(e1372); daf-18(ok480) arIs51[cdh-3::gfp]; arEx2399[daf-18(+)] gravid adult hermaphrodites to lay eggs at 25°C for 7-17 hours, and scored after 1-2 days in dauer (72-89 hours after egg lay). Dauer arrest was confirmed in all mosaics scored by SDS selection (see above). Additional mosaics were identified as L1 larvae to facilitate recognition of myo-3p::mCherry expression patterns resulting from particular lineage losses. Somatic gonadal progression was assessed by arIs51[cdh-3::gfp] expression and germline progression by anatomy.

daf-18 tissue-specific rescue experiments

We used tissue-specific promoters to drive daf-18(+) as part of our analysis of the cellular focus for daf-18 activity. The promoters ckb-3p [21] and hlh-12p [71] have been described in continuous development; we corroborated that they have similar expression in dauer (see below).

The rescuing transgenes were generated in the form promoter.:S::daf-18::T2A::tagBFP2: the viral T2A peptide triggers “ribosomal pausing” such that a single transcript produces two independent proteins (here DAF-18 and TagBFP2) [51], the synthetic intron “S” increases expression efficiency [see below; [55, 56]] and the unc-54 3’UTR was used as a neutral 3’UTR [72, 73]. Rescue was assessed in strains of genotype transgene; daf-7(e1372); daf-18(ok480) arIs51[cdh-3::gfp].

For each promoter, a single-copy insertion transgene is shown in Figure 2B. These were generated and mapped using the standard miniMos protocol [74].

Extrachromosomal arrays were also generated with these constructs and the rescue data are shown in Supplemental Figure S2A. Complex arrays were generated by injecting Spel-digested pCT17(hlh-12p::S::daf-18::T2A::tagBFP2) or pCT19(ckb-3p::S::daf-18(+)::T2A::tagBFP2) at 2 ng/μl along with coinjection marker pCW2(ceh-22p::gfp) (expressed specifically in pharyngeal muscle) digested with Scal at 1 ng/pl and with N2 genomic DNA digested with Pvull at 50ng/μl into GS8925 daf-7(e1372); daf-18(ok480) arIs51[cdh-3::gfp] . F2 progeny with GFP in pharyngeal muscle were isolated to establish independent lines.

ckb-3p-driven somatic gonad rescuing transgenes

Single-copy insertion (Figure 2): arTi195 [ckb-3p::daf-18::T2A::tagBFP2]. arTi195 maps to V.2.22 in an intron of the gene unc-41. Extrachromosomal arrays (Figure S2): arEx2413-arEx2415 [ckb-3p::daf-18::T2A::tagBFP2].

As it was difficult to visualize TagBFP2 from these transgenes to confirm that expression in dauer is similar to that reported in continuous development, we examined arTi112[ckb-3p::mCherry-h2b], in which H2B appears to stabilize the fluorescent protein. mCherry-H2B was observed in the somatic gonad precursors Z1 and Z4 and in all of their descendants in dauer larvae (Figure 1B) and in only three additional cells, tentatively identified as the three MS-derived pharyngeal muscle segment pm6 cells, in dauer larvae as well as in continuous development. Expression was not seen in any other cells in any other stages.

hlh-12p-driven DTC rescuing transgenes

hlh-12p expression is restricted to the DTCs in continuous development from L2 onwards [71]. We verified the specificity in dauer for arEx2416 here.

Single-copy insertion (Figure 2): arTi118 [hlh-12p::daf-18::T2A::tagBFP2], inserted at II:-9.30 in the second exon of irld-65. tagBFP2 expression is too dim to score reliably from this transgene.

Extrachromosomal array: arEx2416 [hlh-12p::daf-18::T2A::tagBFP2] has robust tagBFP2 expression in the DTCs but was not visible in SGBs or AC in dauer (Figure 2C). tagBFP2 was visible only in the DTCs but not SGBs or the AC in 61/61 daf-18(0) dauers, and no non-gonadal expression was observed.

Expression of Cre from both the ckb-3p and hlh-12p promoters using recombination readout reporters (described below) agreed with these observations. Neither of these promoters drives germline expression in any stage, also supported by lack of Cre-mediated excision in the germline (see below).

Extrachromosomal arrays expressing daf-18(+) in specific non-gonadal tissues

Strains with extrachromosomal arrays expressing daf-18(+) cDNA specifically in the intestine (veEx349[ges-1p::daf-18cDNA::myc::daf-18 3’UTRJ) and neurons (veEx344[rgef-1p::daf-18cDNA::myc::daf-18 3’UTR]), as well as from its native promoter (tdEx349[daf-18p::daf-18cDNA::myc::daf-18 3’UTR]), are described in [75] and were kindly provided by Masamitsu Fukuyama and Ann Rougvie. All three arrays express functional DAF-18 based on rescue of a daf-18(0) phenotype in L1 larvae [75] and, in our hands, improved non-gonadal dauer-defective phenotypes such as SDS resistance of daf-18(0) dauers. tdEx349[daf-18p] rescued all morphological defects of daf-18(0) dauer larvae (data not shown). Strains scored were of genotype Ex; daf-7(e3172); daf-18(ok480) arIs51[cdh-3::gfp]. tdEx239 may have integrated spontaneously as it segregated in our crosses in a Mendelian ratio.

Obtaining and scoring dauer larvae for tissue-specific rescue experiments

For arTi195 [ckb-3p::daf-18(+)] and arTi118[hlh-12p::daf-18(+)] single-copy insertion transgenes and extrachromosomal arrays veEx349, veEx344, and tdEx349, 1-day dauers were obtained and SDS-selected by the standard method described above. For other extrachromosomal arrays, 1 day dauers were obtained after an egg-lay instead of using a bleaching protocol to obtain a synchronized population, followed by SDS-selection to obtain dauers. SGB progression was assessed by arIs51[cdh-3::gfp] expression and GSC progression by microscopy as described above.

Cre-mediated excision mosaic experiments

Generation of the loxP>daf-18>loxP single-copy insertion transgene arTi179

arTi179[daf-18p::loxP::daf-18::loxP::GFP::H2B], designated loxP>daf-18>loxP, is a single-copy insertion generated using the standard miniMos protocol [74]. Insertion of arTi179 into I:4.97 in the intergenic region between F25D7.7 and mgl-2 was identified using the standard protocol for mapping miniMos insertions [74].

arTi179 was created from plasmid pCT33. This plasmid contains the entire daf-18(+) genomic region, including introns, starting with the ATG and extending 20 base pairs into the 3’ gene eri-1, with loxP sites positioned to allow for excision of daf-18(+).

We represent the full loxP>daf-18>loxP sequence of pCT33 and arTi179 as follows: daf-18p::SbfIPacI::loxP::aa::daf-18(+)::loxP::PacI::S::GFP::his-58::unc-54 3’UTR, and contains the following elements.

daf-18p is the 1021 bp 5’ flanking region from the upstream gene to the ATG of daf-18.
“aa” denotes base pairs added that, along with daf-18 native 5’ UTR, creates a Kozak sequence [56].
“daf-18(+)” is the genomic sequence of daf-18 including introns, starting with the ATG and extending 20 base pairs into the 3’ gene eri-1. [A one base-pair mutation within the 5th intron (a3989g) does not interfere with rescue of daf-18(0) (see below).
“S” designates a synthetic intron cassette that includes common features of C. elegans introns and increases efficiency of expression [55], and the version used here also includes a Kozak sequence [56].
a his-58 histone tag was included to improve detection of GFP fluorescence.
the unc-54 3’UTR was used as a neutral 3’UTR [72, 73].

arTi179 was designed such that GFP expression is only expected with Cre-mediated recombination. GFP expression was never detected in dauer larvae except in the presence of Cre drivers, and thus tissue-specific excision of daf-18 as monitored by GFP expression was readily assessed in dauer (see below). Although effective for dauer larvae, we note that in continuous development, GFP expression cannot be used to monitor tissue-specific excision because there is a low-level broad GFP expression from arTi179 in all stages of continuous development even in the absence of Cre drivers, likely due to the presence of an SL2 splice acceptor in the daf-18 3’ UTR as per its position as the upstream gene in an operon with eri-1.

Generation of single-copy insertion CRE drivers

We used the Cre recombinase optimized via several modifications for efficiency in C. elegans by [25]. miniMos-based single-copy insertion transgenes arTi168[hlh-12p::Cre], arTi236[ckb-3p::Cre], and arTi235[hlh-1p::Cre] were modeled on constructs use for optimized Cre-expressing transgenes described in [25].

These driver constructs are all of the form promoter.:S::Cre::tbb-2 3’UTR, where “S” designates a synthetic intron cassette that includes a Kozak sequence as above [55, 56]. Each insert was incorporated into the miniMos vector pCFJ910 [74]. arTi168[hlh-12p::CRE(opti)] was mapped to X:24.06 in a noncoding region. arTi179 and arTi235 were not physically mapped but insertion is inferred based on homozygosity and Mendelian behavior during strain constructions.

In pCT37, promoter = hlh-12p and in pCT43, promoter = ckb-3p as used for tissue-specific rescue. In addition, pCT42, promoter = hlh-1p, a 3130 bp region analyzed by [52, 53]. Excision events caused by the Cre driver transgenes were examined in dauer larvae via GFP expression from arTi179[loxP>daf-18>loxP] and an independent Cre readout reporter, heIs105 [25], as described in the next section.

Testing the Cre drivers using heIs105

In the absence of Cre driver-induced excision, the single-copy miniMos insertion heIs105[rps-27::loxP::nls::mCherry::let-858 3’ UTR::loxP::nls::GFP::let-858 3’ UTR] expresses mCherry ubiquitously but not GFP; upon Cre-mediated excision, it expresses GFP but not mCherry [25].

We assessed the patterns of Cre-mediated excision in the presence of our drivers using the heIs105 reporter in dauers formed by starvation at 25° C and in continuously-developing L2 larvae prepared by timed egg lay at 25° C. GFP expression resulting from the hlh-12p and ckb-3p drivers using the heIs105 reporter was broader than expected. We catalogue these differences here, but importantly, they do not change our conclusions about the cellular focus of daf-18. We speculate that the differences reflect the low-level, transient expression of Cre at an early point of a lineage, such that excision occurring early in a lineage as a result of transient expression of Cre is inherited in all lineal descendants even if there is no expression from the promoter in those cells thereafter, while rescue experiments likely require stronger or sustained expression at the time and place of action, as would visualization of the fluorescent reporters generally used to characterize the promoters.

arTi168[hlh-12p::Cre] with heIs105: in addition to the expected DTCs, we observed GFP expression in three cells in the tail, some pharyngeal muscle cells, and some cells of the excretory system. Importantly, no proximal gonad excision was observed (n =7 dauer, n=10 L2). Furthermore, ectopic Cre excision mediated by this driver does not affect the interpretation that the DTCs are not required for SGB and GSC quiescence in dauer larvae, since excision of arTi179[loxP>daf-18>loxP] using this driver in cells did not cause SGB and GSC progression.

arTi236[ckb-3p::Cre] with heIs105 (n=10 dauer, n=10 L2): in addition to the expected expression of GFP in all somatic gonad cells, we also observed expression in some cells of the body wall muscle, pharynx, and tail. This excision pattern is consistent with expression of the driver earlier than expected in the MS lineage. We also observed expression in an occasional intestinal or hypodermal cell.

arTi235[hlh-1p::Cre] and heIs105 (n=10 dauer, n=10 L2): in this case, the observed pattern of excision was as expected. We always observed expression in all body wall muscle cells, where hlh-1 is a main muscle determinant [76] and in many pharyngeal cells and sometimes in the somatic gonad cells as expected from the reported transient expression in MS granddaughters [52, 53]. We were able to exploit this variability for somatic gonad excision to support the conclusion that daf-18 acts in the somatic gonad (Figure 3B).

We also note that we never saw evidence for germline excision caused by these drivers. For both heIs105 and arTi179 [loxP>daf-18>loxP], we examined progeny of hermaphrodites for ubiquitous somatic GFP expression that would have been indicative of a rearrangement in the parent germline. For arTi179 [loxP>daf-18>loxP], we also directly inspected the germline of excision mosaics for GFP expression.

Obtaining tissue-specific excision mosaic dauer larvae

Strains of genotype daf-7(e1372); daf-18(ok480); p::Cre were generated for each Cre driver used. As males, and some hermaphrodites, of genotype arTi179[loxP>daf-18(+)>loxP]; daf-7(e1372); daf-18(ok480); arTi112[ckb03p::mCheny::H2B] constitutively become dauers even at the permissive temperature, we moved dauer larvae to fresh plates at 15°C and used the recovered, post-dauer males for the cross strategy and to maintain the strain.

The strategy shown in Figure 2 was used: daf-18(0) males homozygous for loxP>daf-18(+)>loxP were mated to daf-18(0); hermaphrodites carrying a Cre driver (Figure 3A), ensuring that daf-18(0) F1 hermaphrodite progeny that were scored had no maternal contribution from loxP>daf-18(+)>loxP. This strategy also ensured that the daf-18(0) F1 progeny were heterozygous for loxP>daf-18(+)>loxP, so that only one excision event would be necessary to generate a daf-18(0) excision mosaic; since Cre-mediated recombination of loxP>daf-18(+)>loxP results in daf-18p::loxP::GFP::H2B, cellular expression of GFP-H2B in larvae heterozygous for loxP>daf-18(+)>loxP indicates complete excision and loss of daf-18(+) in that cell.

Crosses were initiated with twelve L4 loxP>daf-18(+)>loxP; daf-18(0) and four L4 daf-18(0); p::Cre hermaphrodites at 20° C. The parent crosses were moved to new plates periodically over 2-3 days and each egg lay was shifted to 25Ό once the parents were removed.

Scoring tissue-specific excision mosaic dauer larvae

Dauers were selected by incubation in 1% SDS and scored 65-100 hours after egg lay, which corresponds to 1-2 days after dauer formation [65]. Only hermaphrodite dauers were scored. As per the strategy above and in Figure 3A, expression of GFP indicates an excision mosaic lacking daf-18(+) in the GFP-expressing cell. Somatic gonad cells were marked with arTi112[ckb-3p:: mCherry-H2B]. SGB progression was scored by increased numbers of cells with mCherry::H2B and confirmed by anatomy, and GSC progression was scored by anatomy. Individual observations of somatic gonad excision of loxP>daf-18(+)>loxP in dauer by driver are:

arTi236[ckb-3p::Cre]: GFP was expressed in cells of the proximal somatic gonad and the DTCs. In addition to implicating the somatic gonad as the cellular focus, the observation that the daf-18 promoter in daf-18p:: loxP::gfp drives expression of GFP in the somatic gonad indicates that the daf-18 gene is expressed in the dauer somatic gonad.

arTi235[hlh-1p::Cre]: Two classes of excision mosaics were identified: (1) individuals that did not express GFP in the somatic gonad even when consistent GFP was seen in the entire body wall muscle (n = 18 in Figure 3A), and (2) individuals that expressed GFP in the somatic gonad as well as the body wall muscle (n = 7 in Figure 3A). That excision mosaics of class 1 do not display SGB and GSC progression, while excision mosaics of class 2 do, strongly supports the conclusion that the somatic gonad is the cellular focus of daf-18 for maintaining quiescence of these blast cells in dauer larvae.

arTi168[hlh-12p::Cre] GFP is expressed in the DTCs but not the proximal gonad. GFP expression was observed in both DTCs in 11/13 dauers and in at least one DTC in the remaining 2 dauers, where autofluorescence may have obscured the second DTC. SGB progression was scored by increased numbers of cells with mCherry::H2B and confirmed by anatomy, and GSC progression was scored by anatomy.

Generating daf-18(0) mosaics in a daf-2(e1370) background

Strain GS9117 daf-2(e1370); daf-18(ok480) arls51[cdh-3::gfp]; arEx2399[daf-18(+)] was used to generate dauers retaining or lacking daf-18(0) in the MS lineage (Figure 4) as described above. Since daf-18(0) fully suppresses the dauer constitutive phenotype of daf-2, the ability to form dauer larvae is one indication that the array rescues daf-18(0). In addition, non-mosaic individuals formed dauer larvae that displayed radial body constriction, pharyngeal constriction, and somatic gonad and germline quiescence (n=7). Mosaic dauers were obtained from 40 GS9117 daf-2(e1370); daf-18(ok480) arls51[cdh-3::gfp]; arEx2399[daf-18(+)] gravid hermaphrodites carrying the array grown at 15°C th at laid eggs at 25°C for approximately 24 hours. Dauers were scored after approximately 1-2 days in dauer (65-101 hours after eggs were laid at 25°C); selection and scoring criteria were conducted as described above for mosaic analysis of GS8095 dauers.

We scored specific lineage losses of arEx2399 in each daf-2(e1370); daf-18(ok480) arIs51[cdh-3::gfp] dauer and grouped simple (lost in one founder cell lineage) and complex (more than one founder cell lineage) losses based on retention or loss of arEx2399[daf-18(+)] in the MS lineage (Figure 4). These data are represented alongside the mosaic data from GS8095 daf-7(e1372); daf-18(ok480) arIs51[cdh-3::gfp]; arEx2399[daf-18(+)] (see above) analyzed the same way. Specific lineage losses are shown in Figure S4. Three mosaic dauers had simple losses in P₂ [germline therefore inferred to be daf-18(0)]: 0/3 had SGB or GSC progression. In 2/2 simple MS losses, both the SGBs and GSCs progressed. Thus, mosaic analysis of daf-18 in both the daf-2 and daf-7 backgrounds indicate a cellular focus for daf-18(0) within the MS lineage for regulating SGB and GSC progression, indicating cell non-autonomy for GSC progression.

Scoring SM progression in daf-18(0) and daf-18(+) expression mosaics

To assess SM progression in daf-7; daf-18(0) dauers, SM descendants were fluorescently marked with arTi133[hlh-8p::ERK-KTRmClover::T2A::mCherry::H2B] (abbreviated in this text as arTi133[hlh-8p::mCherry::H2B], de la Cova et al. 2017). In all control dauers, the two SMs are positioned lateral to the center of the gonad and do not divide (n=24, Figure 4B), but the SMs progressed in 7/7 daf-18(0) dauers (P ≤ 0.001), evident by 8-16 mCherry::H2B-expressing cells in two clusters lateral to the central gonad.

To assess SM progression when SGB and GSC quiescence was rescued, we examined strain GS9083 arTi133[hlh-8p::ERK-KTRmaover::T2A::mCherry::H2B]; daf-7(e1372); daf-18(ok480) arIs51[cdh-3::gfp]; arTi195[ckb-3p::daf-18(+)::T2A::tagBFP2]. SMs were scored as progressing if more than two mCherry-expressing cells were visible in the gonad region, and in 19/20 scored, ≥seven SM descendants were observed and 1/20 had 3 marked cells. In these dauers, SGB quiescence was confirmed by arIs51[cdh-3::gfp] expression restriction to the AC and GSC with anatomy as described above.

Genetic ablation of AC and DTCs by hlh-2(RNAi)

The DTCs and AC can be genetically ablated by feeding hlh-2(RNAi) to L1 larvae [26, 27] (Figure S3). The lag-2 reporter arIs131[lag-2p::2Xnls-yfp] [77] marks the specified DTCs and AC, so absence of YFP in both DTCs and the AC, as well as abrogation of gonad arm extension indicate successful DTC and AC “ablation”. SGB divisions were scored using arTi112[ckb-3p::mCherry::H2B] and germline progression by anatomy.

Post-dauer phenotypes of gonad-specific excision mosaics

After tissue-specific excision of loxP>daf-18(+)>loxP (Figure 4), 1-2 day dauers were obtained by SDS selection, placed on individual plates containing E. coli, and allowed to recover at the permissive temperature, 15° C. Post-dauer adult hermaphrodites were scored for sterility and egg-laying defects. Sterility would be a direct effect of aberrant somatic gonad and/or germline development.

Egg-laying defects, evident by the presence of live larvae inside the adult hermaphrodite from egg retention and hatching, may result from abnormal development of the somatic gonad, vulva, vulval-uterine connection, or reproductive muscles.

QUANTIFICATION AND STATISTICAL ANALYSIS

When comparing two genotypes for the frequency of two outcomes, a two-tailed 2×2 Fisher’s exact test was used. Differences were considered significant if the p-value is less than or equal to 0.05, and specific p-values for each comparison are given in the figure legends or main text.

Supplementary Material

NIHMS1520543-supplement-1.pdf^{(532.5KB, pdf)}

Highlights.

C. elegans dauer diapause is a state of global and reversible developmental arrest
Somatic gonad blast cells (SGB) and germline stem cells (GSC) are quiescent
daf-18/PTEN acts in the somatic gonad to maintain quiescence of SGBs and GSCs
daf-18 regulates a signal to synchronize somatic gonad and germline development

Acknowledgments

We gratefully acknowledge much valuable advice, discussion and comments on the manuscript from Xantha Karp. We also give special thanks to Michelle Attner for very helpful guidance and comments on the manuscript, and to Maria Sallee, Claire de la Cova and Katherine Luo for discussion at various stages of this project. We also thank Justin Shaffer, Hana Littleford, and Bryan Wang for help in constructing some strains, Dan Shaye for the glo-1 E lineage marker, Masamitsu Fukuyama and Ann Rougvie for strains, and all members of the Greenwald lab for advice and encouragement. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Research reported in this publication was entirely supported by the Institute of General Medicine of the National Institutes of Health under award numbers R01GM115718 (to I.G.) and F31GM116508 (to C.C.T.). C.C.T. was also supported by NIH NIGMS MSTP Training Grant T32GM007367. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Footnotes

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Declaration of Interests

The authors declare no competing interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS1520543-supplement-1.pdf^{(532.5KB, pdf)}

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PERMALINK

Cell non-autonomous function of daf-18/PTEN in the somatic gonad coordinates somatic gonad and germline development in C. elegans dauer larvae

Claudia C Tenen

Iva Greenwald

Summary

eTOC

Results

daf-18 activity prevents developmental progression of the somatic gonad in dauer larvae

Figure 1. Developmental progression of Somatic Gonad Blast cells (SGBs) and Germline Stem Cells (GSCs) in daf-18(0) dauers.

daf-18 functions in the somatic gonad primordium to prevent SGB and GSC developmental progression in dauer

Figure 2. daf-18 acts in the somatic gonad primordium to maintain SGB and GSC quiescence in dauer.

daf-18 has a diffuse cellular focus within the somatic gonad

Figure 3. daf-18 has a diffuse cellular focus within the somatic gonad.

Loss of daf-18 permits SGB and GSC progression in other dauer-constitutive mutant backgrounds

Figure 4. Evidence for a novel signal regulated by daf-18/PTEN and model.

The somatic gonad does not regulate sex myoblast quiescence in dauer larvae

Post-dauer defects in daf-18 somatic gonad excision mosaics reveal the importance of maintaining developmental quiescence in dauer

Discussion

STAR Methods

CONTACT FOR REAGENT AND RESOURCE SHARING

EXPERIMENTAL MODEL AND SUBJECT DETAILS

C. elegans strains and transgenes

METHOD DETAILS

Identifying and scoring dauer larvae

Imaging

Evaluating SGB progression using fluorescent markers

Scoring GSC quiescence and progression in daf-7(e1372) or daf-2(e1370) dauers

Scoring GSC quiescence and progression in daf-9(dh6) dauers

daf-18 mosaic analysis for cellular focus

Generation of arEx2399[daf-18(+)] for mosaic analysis

Generation of daf-18(0) mosaic dauers

daf-18 tissue-specific rescue experiments

ckb-3p-driven somatic gonad rescuing transgenes

hlh-12p-driven DTC rescuing transgenes

Extrachromosomal arrays expressing daf-18(+) in specific non-gonadal tissues

Obtaining and scoring dauer larvae for tissue-specific rescue experiments

Cre-mediated excision mosaic experiments

Generation of the loxP>daf-18>loxP single-copy insertion transgene arTi179

Generation of single-copy insertion CRE drivers

Testing the Cre drivers using heIs105

Obtaining tissue-specific excision mosaic dauer larvae

Scoring tissue-specific excision mosaic dauer larvae

Generating daf-18(0) mosaics in a daf-2(e1370) background

Scoring SM progression in daf-18(0) and daf-18(+) expression mosaics

Genetic ablation of AC and DTCs by hlh-2(RNAi)

Post-dauer phenotypes of gonad-specific excision mosaics

QUANTIFICATION AND STATISTICAL ANALYSIS

Supplementary Material

Highlights.

Acknowledgments

Footnotes

References

Associated Data

Supplementary Materials

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