A transitory signaling center controls timing of primordial germ cell differentiation

Abstract

Organogenesis requires exquisite spatiotemporal coordination of cell morphogenesis, migration, proliferation, and differentiation of multiple cell types. For gonads, this involves complex interactions between somatic and germline tissues. During Drosophila ovary morphogenesis, primordial germ cells (PGCs) either are sequestered in stem cell niches and are maintained in an undifferentiated germline stem cell state or transition directly toward differentiation. Here, we identify a mechanism that links hormonal triggers of somatic tissue morphogenesis with PGC differentiation. An early ecdysone pulse initiates somatic swarm cell (SwC) migration, positioning these cells close to PGCs. A second hormone peak activates Torso-like signal in SwCs, which stimulates the Torso receptor tyrosine kinase (RTK) signaling pathway in PGCs promoting their differentiation by de-repression of the differentiation gene, bag of marbles. Thus, systemic temporal cues generate a transitory signaling center that coordinates ovarian morphogenesis with stem cell self-renewal and differentiation programs, highlighting a more general role for such centers in reproductive and developmental biology.

Keywords: Drosophila; Torso; ecdysone; germline; gonad morphogensis; primordial germ cells; stem cells; steroid hormone; swarm cell; transitory signaling center.

Figure 1. SwC morphogenesis establishes a posterior gonad domain

(A) Schematic of larval ovary development. At EL3 (72h AEL), primordial germ cells (PGCs - light green) proliferate and are in close contact with intermingled cells (ICs - red) and follicle stem cell progenitors (FSCPs - dark blue). Terminal filament progenitors (TFs - orange) and sheath cells (SH - light blue) are specified at the anterior; swarm cells (SwCs - purple) are located anterolateral. An early ecdysone peak (~90h AEL) induces niche formation, SwCs are located dorsolateral. A late ecdysone peak (~100h AEL) initiates PGC differentiation (dark green); whereas PGCs close to the forming niches (TFs and cap cells (CC) - yellow) are maintained as germline stem cells (GSCs); SwCs form a posterior domain. (B) Violin plot from scRNA-seq. data for sim; gene expression levels (y axis) for each ovarian cell cluster (x axis) are given, each dot represents a cell. (C-E) Anti-Sim antibody detects SwCs (orange outline) during development. TF morphogenesis (arrowheads) marked by anti-Engrailed (En) antibody (white) marks developmental stages; PGC compartment is green outlined. (F) Spatial relationship of PGCs (vasa), FSCPs (bond) and SwCs (sim), detected by HCR in-situ hybridization at LL3. Scale bars indicate 10 μm. See also Figure S1.

Figure 2. Lineage tracing reveals transitory nature of SwCs

(A-H) sim-Gal4 driven expression of G-TRACE follows SwC fate: current (RFP in magenta) and lineage expression (GFP in green); SwC locations and PGC positions are marked by orange and green outline, respectively. (A-G) Current expression reveals sim-Gal4 activity in SwCs from L2 on, recapitulating SwC morphogenesis. In pupae, by 60h APF current expression can no longer be detected. (E-F) At 36h-60h APF, SwC descendants are found at the posterior separated from PGCs (green outline) by follicle cells labeled with anti-Fasciclin3 antibodies (Fas3) (blue bracket). (F) By 60h APF SwC lineage contributes to the calyx (blue outlined); few lineage-labeled cells can be found in the developing peritoneal sheath (white arrowheads). (F-G) SwCs at the base of developing ovarioles (yellow arrowheads in insets; asterisk and white outline mark extra-ovariolar cavity in F) undergo apoptosis marked by anti-Dcp-1 antibodies (G). (H) Larval SwCs descendants were detected in the peritoneal sheath (Phalloidin, arrowheads) of adults. Scale bar indicates 10 μm, except 100 μm in H. See also Figure S1.

Figure 3. Ecdysone pulses promote SwC morphogenesis and PGC differentiation

(A-D) Effect of EcR^DN expression on SwC morphogenesis. SwCs are labeled with anti-Sim antibodies (orange outline); all cell outlines and the fusome in PGCs are marked by anti-α-Spectrin. (A) SwCs occupy the posterior in control ovaries at LL3; (B) pan-somatic (tj-Gal4) or (C and D) SwCs specific (sim-Gal4, cv-2-Gal4) expression of EcR^DN impacts SwC morphogenesis. (E-F) Eb1:GFP marks SwC protrusions and DAPI marks nuclear morphology. (E-E’) Wildtype SwCs initiate posterior migration shortly after ML3, indicated by directional protrusions and elongated nuclear morphology (arrowheads and outline). (F) SwCs expressing EcR^DN lack motile behavior. (G) Graphical representation of SwC motility index in control and EcR^DN expressing SwCs. (H-J) PGC differentiation is monitored by bamP:GFP, PGC domain is outlined in green. (H) Wildtype PGCs initiate differentiation at LL3; (I-J) bamP:GFP reporter expression is absent in EcR^DN expressing SwCs (note, bamP:GFP reporter shows ectopic expression in TFs). (K) Graph depicting relative bamP:GFP expression levels as exemplified in H-J. Each data point represents a single PGC. Error bars represent SD. Scale bars indicate 10 μm. See also Figure S2.

Figure 4. SwCs express tsl to initiate PGC differentiation in response to a late larval ecdysone pulse

(A) Violin plot from scRNA-seq. data for tsl; gene expression levels (y axis) for each ovarian cell cluster (x axis) are given, each dot represents a cell. (B-D) PGC differentiation status is indicated by bamP:GFP expression; PGC domain is outlined in green. (B) PGCs in control ovaries express bamP:GFP. In tsl-RNAi knock-down (C) bamP:GFP expression was decreased, in tsf^)E (D) bamP:GFP expression was increased. Precocious PGC differentiation is highlighted by presence of branched fusomes (anti-a-Spectrin, inset). (E) Graph quantifying bamP:GFP expression levels as exemplified in B-D. Each data point represents a single PGC. (F, F’) HCR in-situ hybridization for tsl and sim mRNAs at LL3, germ cells are labeled with anti-Vasa antibodies. (G) Graph showing average number of tsl mRNA foci per SwC at EL3 to LL3; each data-point represents a single ovary. (H-J) tsl-Gal4 driven expression of G-TRACE. No expression was detected at EL3, few SwCs were labeled at ML3 and many were labeled at LL3. (K) qPCR measurements showing increased tsl mRNA levels from ML3 to LL3 in wildtype, but not when EcR^DN was expressed in soma. (L-N) SwCs are marked via anti-Sim antibodies (orange outline), PGC differentiation status is indicated by bamP:GFP (PGC domain outlined in green). (L-M) tsl-Gal4 driven EcR^DN: SwC migration was unaffected but PGC differentiation was blocked. (N) Reintroduction of tsl rescued the EcR^DN phenotype. (O) Graph depicting relative bamP:GFP expression levels as exemplified in L-N. Each data point represents a single PGC. Error bars represent SD. Scale bar indicates 10 μm. See also Figure S2.

Figure 5. Temporal control of Tsl expression in SwCs promotes PGCs differentiation via RTK Torso signaling

(A) Schematic summary of SwC-to-PGC signaling via Tsl-Torso pathway. (B) HCR in-situ hybridizations for torso, trk, tll and vasa mRNAs. (C-H) PGC differentiation status is indicated by bamP:GFP; PGC domain is outlined in green. RNAi-mediated knock-down of torso (D), trk (E), or tll (F), resulted in decreased bamP:GFP expression when compared to control. Expression of an activated version of trk (trk^[C108]) (G), and overexpression of tll (H), resulted in precocious PGC differentiation, highlighted by presence of branched fusomes (anti-a-Spectrin, inset). (I) Graph depicting relative bamP:GFP expression levels as exemplified in C-H. Each data point represents a single PGC; P values in relation to control. Error bars represent SD. Scale bar indicates 10 μm. See also Figure S3.

Figure 6. Torso pathway promotes PGCs differentiation by alleviating Krüppel-mediated repression of bam promoter activity.

(A-C) bamP:GFP expression indicates PGC differentiation status, PGC domain is outlined in green. (B) PGC specific Kr-RNAi knockdown results in precocious differentiation highlighted by presence of branched fusomes (anti-a-Spectrin, inset), (C) Kr blocks differentiation. (D) Graph depicting relative bamP:GFP expression levels as shown in A-C. Each data point represents a single PGC. (E) Measurements of Kr mRNA foci detected by HCR in PGCs at EL3 to LL3; each data-point represents a single ovary. (F) qPCR measurements show decrease of Kr mRNA levels from ML3 to LL3 in wild type gonads but not when tsl-RNAi was expressed in soma. (G) qPCR measurement of bam mRNA levels, showing significant increase from ML3 to LL3 in wildtype. Kr-RNAi in PGCs results in elevated bam levels at ML3 while Kr^OE decreased bam levels at LL3. (H) Schematic representation of bam transcriptional reporters. In bamP^wt:EGFPd2, the bam promoter sequence was fused to a destabilized EGFP; known enhancer elements (orange) and the Mad/Med targeted silencer element (red) are indicated. In bamP^krmut:EGFPd2A a putative Kr binding site (purple) was mutated. (I-J) Reporter expression is increased when the Kr binding site is mutated. (K) Graph depicting relative expression levels of the bam reporters as exemplified in I-J. Each data point represents a single PGC; Error bars represent SD. Scale bar indicates 10 μm. (L) Model for ecdysone control of SwC morphogenesis and PGC signaling. See also Figure S4.