altered in the phosphorylation mutants (*zpg* Y352F and *zpg* Y352F/S356A; B") and three different mutations of extracellular cysteines (*zpg* C6S, *zpg* C145S, *zpg* C236S; B").
(TIF)

**S3 Fig. A single copy of Zpg::GFP GR results in expression levels that are similar to those seen in zpg heterozygous mutant flies.** (A) Fluorescence intensity quantification (n = 12 images measured per genotype; mean fluorescence intensity in arbitrary units) of Zpg anti-body staining in testes of wildtype control, flies heterozygous for the *zpg*<sup>2533</sup> null allele (*zpg*<sup>2533</sup>/+ ) and *zpg* mutants rescued with one copy of the genomic rescue construct (*zpg*::GFP GR). Similar levels of expression are observed in testes of *zpg* mutants rescued with *zpg*::GFP GR and *zpg*<sup>2533</sup> heterozygotes. (B-B') Representative images showing expression levels for wildtype (B) and *zpg* mutants rescued with *zpg*::GFP GR (B'). Hubs are marked by asterisks. Scale bars represent 50 µm. p-values are indicated by asterisks with *p*<0.05, **p<0.01, ***p<0.001.
(TIF)

**S4 Fig. The late germ cell marker Boule is expressed in testes of zpg::GFP GR and phospho mutant flies, indicating normal germ cell differentiation.** (A-D) Testes stained for the meiotic germ cell marker Boule (red) and nuclei (blue). In testes of wildtype flies (A) or from *zpg* mutants rescued with *zpg*::GFP GR (B), which show a complete rescue of the *zpg* null phenotype, Boule signal is found in late germ cell cysts as well as in long and parallel spermatid bundles. In *zpg* null mutants (C), meiotic stages are not reached hence no Boule signal is detected. Testes of both phosphorylation site mutants (*zpg* Y352F in D, *zpg* Y352F/S356A in E) show strong Boule signal and the parallel organization of spermatid bundles. Hubs are marked by asterisks. (F) Testes stained for nuclear marker (white), arrows indicate elongated spermatid bundles. Scale bars represent 50 µm.
(TIF)

**S5 Fig. Strong defects and absence of membrane localization is observed in testes of GFP-tagged zpg cysteine mutants.** (A-L) Immunostaining for germ and somatic cells in wild type (A-C), *zpg* null mutant (D-F), *zpg* null mutant rescued with *zpg*::GFP C6S (G-I) and *zpg* null mutant rescued with *zpg*::GFP C26S (J-L). Both C6S::GFP and C26S::GFP rescue exhibit a phenotype that is indistinguishable from the *zpg* null mutant with a decreased number of germ cells (Vasa+) and late somatic cells (Eya+) as well as an increased number of early-mid somatic cells (Zfh-1+ and Tj+). The immunostainings, as well as the quantification of (M) germline stem cells (GSCs), (N) Zfh-1-positive cells, (O) Tj-positive cells and (P) Eya-positive cells reveals a strong, nearly null-mutant like phenotypes in *zpg*::GFP C6S and *zpg*::GFP C26S rescued testes, indicating a strong loss of function. This is consistent with the absence of the mutated Zpg::GFP C6S and Zpg::GFP C26S proteins from the plasma membrane (S-U). (Q-T) Colocalization of endogenous, unmutated Zpg and GFP-tagged, mutated Zpg in flies with one copy of endogenous Zpg. *zpg* GFP::GR and *zpg* heterozygous controls are depicted in B-B' and C-C', respectively. In testes expressing the *zpg*::GFP C6S (S-S') and *zpg*::GFP C26S (T-T') mutant constructs, the GFP-tagged mutated Zpg accumulates intracellularly, while endogenous Zpg mainly localizes to the plasma membrane. This results in low Pearson colocalization coefficients (U) upon quantification of the colocalization of endogenous and mutated Zpg testes of flies with the *zpg*::GFP C6S and *zpg*::GFP C26S mutant transgenes compared to the *zpg*::GFP GR control.
(TIF)

**S6 Fig. Analysis of Zpg localization and somatic cell development in zpg D50 point mutants.** (A-E) The subcellular localization of mutant Zpg proteins is revealed by staining for GFP (green) as all rescue transgenes contain a GFP tag at the C-terminus of Zpg, Fas3 (red) is
altered in the phosphorylation mutants (*zpg* Y352F and *zpg* Y352F/S356A; B") and three different mutations of extracellular cysteines (*zpg* C6S, *zpg* C145S, *zpg* C236S; B").
(TIF)

**S3 Fig. A single copy of Zpg::GFP GR results in expression levels that are similar to those seen in zpg heterozygous mutant flies.** (A) Fluorescence intensity quantification (n = 12 images measured per genotype; mean fluorescence intensity in arbitrary units) of Zpg anti-body staining in testes of wildtype control, flies heterozygous for the *zpg*<sup>2533</sup> null allele (*zpg*<sup>2533</sup>/+ ) and *zpg* mutants rescued with one copy of the genomic rescue construct (*zpg*::GFP GR). Similar levels of expression are observed in testes of *zpg* mutants rescued with *zpg*::GFP GR and *zpg*<sup>2533</sup> heterozygotes. (B-B') Representative images showing expression levels for wildtype (B) and *zpg* mutants rescued with *zpg*::GFP GR (B'). Hubs are marked by asterisks. Scale bars represent 50 µm. p-values are indicated by asterisks with *p*<0.05, **p<0.01, ***p<0.001.
(TIF)

**S4 Fig. The late germ cell marker Boule is expressed in testes of zpg::GFP GR and phospho mutant flies, indicating normal germ cell differentiation.** (A-D) Testes stained for the meiotic germ cell marker Boule (red) and nuclei (blue). In testes of wildtype flies (A) or from *zpg* mutants rescued with *zpg*::GFP GR (B), which show a complete rescue of the *zpg* null phenotype, Boule signal is found in late germ cell cysts as well as in long and parallel spermatid bundles. In *zpg* null mutants (C), meiotic stages are not reached hence no Boule signal is detected. Testes of both phosphorylation site mutants (*zpg* Y352F in D, *zpg* Y352F/S356A in E) show strong Boule signal and the parallel organization of spermatid bundles. Hubs are marked by asterisks. (F) Testes stained for nuclear marker (white), arrows indicate elongated spermatid bundles. Scale bars represent 50 µm.
(TIF)

**S5 Fig. Strong defects and absence of membrane localization is observed in testes of GFP-tagged zpg cysteine mutants.** (A-L) Immunostaining for germ and somatic cells in wild type (A-C), *zpg* null mutant (D-F), *zpg* null mutant rescued with *zpg*::GFP C6S (G-I) and *zpg* null mutant rescued with *zpg*::GFP C26S (J-L). Both C6S::GFP and C26S::GFP rescue exhibit a phenotype that is indistinguishable from the *zpg* null mutant with a decreased number of germ cells (Vasa+) and late somatic cells (Eya+) as well as an increased number of early-mid somatic cells (Zfh-1+ and Tj+). The immunostainings, as well as the quantification of (M) germline stem cells (GSCs), (N) Zfh-1-positive cells, (O) Tj-positive cells and (P) Eya-positive cells reveals a strong, nearly null-mutant like phenotypes in *zpg*::GFP C6S and *zpg*::GFP C26S rescued testes, indicating a strong loss of function. This is consistent with the absence of the mutated Zpg::GFP C6S and Zpg::GFP C26S proteins from the plasma membrane (S-U). (Q-T) Colocalization of endogenous, unmutated Zpg and GFP-tagged, mutated Zpg in flies with one copy of endogenous Zpg. *zpg* GFP::GR and *zpg* heterozygous controls are depicted in B-B' and C-C', respectively. In testes expressing the *zpg*::GFP C6S (S-S') and *zpg*::GFP C26S (T-T') mutant constructs, the GFP-tagged mutated Zpg accumulates intracellularly, while endogenous Zpg mainly localizes to the plasma membrane. This results in low Pearson colocalization coefficients (U) upon quantification of the colocalization of endogenous and mutated Zpg testes of flies with the *zpg*::GFP C6S and *zpg*::GFP C26S mutant transgenes compared to the *zpg*::GFP GR control.
(TIF)

**S6 Fig. Analysis of Zpg localization and somatic cell development in zpg D50 point mutants.** (A-E) The subcellular localization of mutant Zpg proteins is revealed by staining for GFP (green) as all rescue transgenes contain a GFP tag at the C-terminus of Zpg, Fas3 (red) is
used to mark the hub and nuclei are stained with DAPI (blue). GFP single channel is depicted in white (A'-E'). zpg::GFP GR rescue control (A-A') shows GFP localization at the membrane. Zpg null mutants (B-B') do not express GFP. In testes of zpg mutants rescued with the zpg D50A (C-C') or zpg D50R (D-D') rescue constructs, which show mild germ cell differentiation defects, the GFP signal is strongly enriched at the plasma membrane, indicating normal localization of the mutant GJ proteins. The low number of germ cells in testes of zpg mutants rescued with the zpg D50K mutant rescue construct (E-E') makes it hard to determine the localization of the GFP-tagged mutant Zpg protein. Therefore, localization of this mutant was analyzed in a heterozygous background, containing one copy of the endogenous zpg, this data is shown in Fig 5. (F-T) Analysis of germ cell and somatic cell markers in the N-terminal mutant rescue constructs. Staining for Vasa (mitotic germ cells, left and right panel; green) and the somatic markers Zfh-1 (early soma, left panel; grey), Tj (early-mid soma, middle panel; magenta) and Eya (late soma, right panel; magenta). Hubs are marked with Fas3 in grey, nuclei are labelled in blue. Wild type control depicted in F-H, zpg null mutant in I-K. The number of early somatic cells (Zfh-1+, Tj+) in the testes of zpg mutants rescued with either zpg D50A (L, M), zpg D50R (O, P) or zpg D50K rescue constructs (R, S). All analyzed mutants have a lower number of Eya+ cells than wt (right panel). Associated quantifications are shown in Fig 5. Hubs are marked with dashes. Scale bars represent 50 µm. (TIF)

**S7 Fig. Analysis of Zpg localization and somatic cell development in zpg D59 point mutants.** (A-E) The subcellular localization of mutant Zpg proteins is revealed by staining for GFP (green) as all rescue transgenes contain a GFP tag at the C-terminus of Zpg, Fas3 (red) is used to mark the hub and nuclei are stained with DAPI (blue). GFP single channel is depicted in white (A'-E'). zpg::GFP GR rescue control (A-A') shows GFP localization at the membrane. zpg null mutants (B-B') do not express GFP. The low number of germ cells in testes of zpg mutants rescued with the zpg D59A mutant rescue construct (E-E') makes it hard to determine the localization of the GFP-tagged mutant Zpg protein. In testes of zpg null mutants rescued with the zpg D59N (D-D') or the zpg D59H (E-E') rescue transgenes, the GFP signal is mainly concentrated at the membrane. Therefore, localization of these mutants was analyzed in a heterozygous background, containing one copy of the endogenous zpg, this data is shown in Fig 6. (F-T) Analysis of germ cell and somatic cell markers in the N-terminal mutants. Staining for Vasa (mitotic germ cells, left and right panel; green) and the somatic markers Zfh-1 (early soma, left panel; grey), Tj (early-mid soma, middle panel; magenta) and Eya (late soma, right panel; magenta). Hubs are marked with Fas3 in grey, nuclei are labelled in blue. Wildtype control is depicted in F-H, zpg null mutant in I-K. In testes of zpg null mutants rescued with the D59A rescue construct a strong somatic cell differentiation defect was seen with elevated cell counts for the early somatic markers Zfh-1 (L) and Tj (M), whereas these cell counts appear wildtype for zpg null mutants rescues with either the zpg D59N (O, P) or zpg D59H (R, S) rescue construct. All analyzed mutants have a lower number of Eya+ cells compared to wt (right panel). Associated quantifications are shown in Fig 6. Hubs are marked with dashes. Scale bars represent 50 µm. (TIF)

**S8 Fig. Analysis of Zpg localization and somatic cell development in N-terminal mutants.** (A-E) The subcellular localization of mutant Zpg proteins is revealed by staining for GFP (green) as all rescue transgenes contain a GFP tag at the C-terminus of Zpg, Fas3 (red) is used to mark the hub and nuclei are stained with DAPI (blue). GFP single channel is depicted in white (A'-E'). zpg::GFP GR rescue control (A-A') shows GFP localization at the membrane. zpg null mutants (B-B') do not express GFP. The low number of germ cells in testes of zpg
used to mark the hub and nuclei are stained with DAPI (blue). GFP single channel is depicted in white (A'-E'). zpg::GFP GR rescue control (A-A') shows GFP localization at the membrane. Zpg null mutants (B-B') do not express GFP. In testes of zpg mutants rescued with the zpg D50A (C-C') or zpg D50R (D-D') rescue constructs, which show mild germ cell differentiation defects, the GFP signal is strongly enriched at the plasma membrane, indicating normal localization of the mutant GJ proteins. The low number of germ cells in testes of zpg mutants rescued with the zpg D50K mutant rescue construct (E-E') makes it hard to determine the localization of the GFP-tagged mutant Zpg protein. Therefore, localization of this mutant was analyzed in a heterozygous background, containing one copy of the endogenous zpg, this data is shown in Fig 5. (F-T) Analysis of germ cell and somatic cell markers in the N-terminal mutant rescue constructs. Staining for Vasa (mitotic germ cells, left and right panel; green) and the somatic markers Zfh-1 (early soma, left panel; grey), Tj (early-mid soma, middle panel; magenta) and Eya (late soma, right panel; magenta). Hubs are marked with Fas3 in grey, nuclei are labelled in blue. Wild type control depicted in F-H, zpg null mutant in I-K. The number of early somatic cells (Zfh-1+, Tj+) in the testes of zpg mutants rescued with either zpg D50A (L, M), zpg D50R (O, P) or zpg D50K rescue constructs (R, S). All analyzed mutants have a lower number of Eya+ cells than wt (right panel). Associated quantifications are shown in Fig 5. Hubs are marked with dashes. Scale bars represent 50 µm. (TIF)

**S7 Fig. Analysis of Zpg localization and somatic cell development in zpg D59 point mutants.** (A-E) The subcellular localization of mutant Zpg proteins is revealed by staining for GFP (green) as all rescue transgenes contain a GFP tag at the C-terminus of Zpg, Fas3 (red) is used to mark the hub and nuclei are stained with DAPI (blue). GFP single channel is depicted in white (A'-E'). zpg::GFP GR rescue control (A-A') shows GFP localization at the membrane. zpg null mutants (B-B') do not express GFP. The low number of germ cells in testes of zpg mutants rescued with the zpg D59A mutant rescue construct (E-E') makes it hard to determine the localization of the GFP-tagged mutant Zpg protein. In testes of zpg null mutants rescued with the zpg D59N (D-D') or the zpg D59H (E-E') rescue transgenes, the GFP signal is mainly concentrated at the membrane. Therefore, localization of these mutants was analyzed in a heterozygous background, containing one copy of the endogenous zpg, this data is shown in Fig 6. (F-T) Analysis of germ cell and somatic cell markers in the N-terminal mutants. Staining for Vasa (mitotic germ cells, left and right panel; green) and the somatic markers Zfh-1 (early soma, left panel; grey), Tj (early-mid soma, middle panel; magenta) and Eya (late soma, right panel; magenta). Hubs are marked with Fas3 in grey, nuclei are labelled in blue. Wildtype control is depicted in F-H, zpg null mutant in I-K. In testes of zpg null mutants rescued with the D59A rescue construct a strong somatic cell differentiation defect was seen with elevated cell counts for the early somatic markers Zfh-1 (L) and Tj (M), whereas these cell counts appear wildtype for zpg null mutants rescues with either the zpg D59N (O, P) or zpg D59H (R, S) rescue construct. All analyzed mutants have a lower number of Eya+ cells compared to wt (right panel). Associated quantifications are shown in Fig 6. Hubs are marked with dashes. Scale bars represent 50 µm. (TIF)

**S8 Fig. Analysis of Zpg localization and somatic cell development in N-terminal mutants.** (A-E) The subcellular localization of mutant Zpg proteins is revealed by staining for GFP (green) as all rescue transgenes contain a GFP tag at the C-terminus of Zpg, Fas3 (red) is used to mark the hub and nuclei are stained with DAPI (blue). GFP single channel is depicted in white (A'-E'). zpg::GFP GR rescue control (A-A') shows GFP localization at the membrane. zpg null mutants (B-B') do not express GFP. The low number of germ cells in testes of zpg
mutants rescued with either the *zpg* D21A (C-C'), *zpg* D21N (D-D'), or *zpg* delta2-5 rescue constructs (E-E') makes it hard to determine the localization of the GFP-tagged mutant Zpg protein. Therefore, localization of these mutants was analysed in a heterozygous background, containing one copy of the endogenous *zpg*, this data is shown in Fig 7. (F-T) Analysis of germ cell and somatic cell markers in the N-terminal mutants. Staining for Vasa (mitotic germ cells, left and right panel; green) and the somatic markers Zfh-1 (early soma, left panel; grey), Tj (early-mid soma, middle panel; magenta) and Eya (late soma, right panel; magenta). Hubs are marked with Fas3 in grey, nuclei are labelled in blue. Wild type control depicted in F-H, *zpg* null mutant in I-K. Testes of *zpg* mutants rescues with the *zpg* D21A (L-N), *zpg* D21N (O-Q) or *zpg* delta2-5 (R-T) mutants show a lower number of Vasa-positive germ cells and Eya-positive late somatic cells, while at the same time the number of early somatic cells (Zfh-1- and Tj-positive) is increased. This phenotype, seen in all three N-terminal mutants is indistinguishable from that of the *zpg* null. Associated quantification are shown in Fig 7. Hubs are marked with dashes. Scale bars represent 50 µm.

(TIF)

**S9 Fig. Colocalization of endogenous and mutant Zpg proteins.** Representative scatter plots generated with the JaCoP plugin in FIJI, one shown per genotype. The signal intensity for endogenous Zpg is plotted against the signal intensity of the GFP-tagged mutated Zpg in flies that have one copy of endogenous and one copy of mutated protein. A linear relationship between endogenous and GFP-tagged signal intensity, like in *zpg::GFP* GR flies (A) indicates strong colocalization of mutated and endogenous Zpg at the membrane. For *zpg* D21A (B) and *zpg* D21N (C) a weak colocalization is detected. Strong colocalization, similar to the control in A, is found in heterozygotes of *zpg* delta2-5 (D), *zpg* D50A (E), *zpg* D50R (F), *zpg* D50K (G), *zpg* D59A (H), *zpg* D59H (I) and *zpg* D59N (J), whereas colocalization is minimal for *zpg* C6S::GFP (K) and *zpg* C26S::GFP (L).

(TIF)

**Author Contributions**

**Conceptualization:** Yanina-Yasmin Pesch, Guy Tanentzapf.

**Data curation:** Yanina-Yasmin Pesch.

**Formal analysis:** Yanina-Yasmin Pesch, Vivien Dang, Fayeza Islam, Priya Kaur, Ciaran R. McFarlane.

**Funding acquisition:** Filip Van Petegem, Guy Tanentzapf.

**Investigation:** Yanina-Yasmin Pesch, Vivien Dang, Fayeza Islam, Priya Kaur, Christopher M. Smendziuk, Ciaran R. McFarlane, Pierre-Yves Musso.

**Methodology:** Michael John Fairchild, Darius Camp, Christopher M. Smendziuk, Anat Messenberg.

**Project administration:** Guy Tanentzapf.

**Resources:** Michael John Fairchild, Darius Camp, Christopher M. Smendziuk, Anat Messenberg.

**Software:** Rosalyn Carr.

**Supervision:** Filip Van Petegem, Guy Tanentzapf.

**Validation:** Yanina-Yasmin Pesch.
mutants rescued with either the *zpg* D21A (C-C'), *zpg* D21N (D-D'), or *zpg* delta2-5 rescue constructs (E-E') makes it hard to determine the localization of the GFP-tagged mutant Zpg protein. Therefore, localization of these mutants was analysed in a heterozygous background, containing one copy of the endogenous *zpg*, this data is shown in Fig 7. (F-T) Analysis of germ cell and somatic cell markers in the N-terminal mutants. Staining for Vasa (mitotic germ cells, left and right panel; green) and the somatic markers Zfh-1 (early soma, left panel; grey), Tj (early-mid soma, middle panel; magenta) and Eya (late soma, right panel; magenta). Hubs are marked with Fas3 in grey, nuclei are labelled in blue. Wild type control depicted in F-H, *zpg* null mutant in I-K. Testes of *zpg* mutants rescues with the *zpg* D21A (L-N), *zpg* D21N (O-Q) or *zpg* delta2-5 (R-T) mutants show a lower number of Vasa-positive germ cells and Eya-positive late somatic cells, while at the same time the number of early somatic cells (Zfh-1- and Tj-positive) is increased. This phenotype, seen in all three N-terminal mutants is indistinguishable from that of the *zpg* null. Associated quantification are shown in Fig 7. Hubs are marked with dashes. Scale bars represent 50 µm.

(TIF)

**S9 Fig. Colocalization of endogenous and mutant Zpg proteins.** Representative scatter plots generated with the JaCoP plugin in FIJI, one shown per genotype. The signal intensity for endogenous Zpg is plotted against the signal intensity of the GFP-tagged mutated Zpg in flies that have one copy of endogenous and one copy of mutated protein. A linear relationship between endogenous and GFP-tagged signal intensity, like in *zpg::GFP* GR flies (A) indicates strong colocalization of mutated and endogenous Zpg at the membrane. For *zpg* D21A (B) and *zpg* D21N (C) a weak colocalization is detected. Strong colocalization, similar to the control in A, is found in heterozygotes of *zpg* delta2-5 (D), *zpg* D50A (E), *zpg* D50R (F), *zpg* D50K (G), *zpg* D59A (H), *zpg* D59H (I) and *zpg* D59N (J), whereas colocalization is minimal for *zpg* C6S::GFP (K) and *zpg* C26S::GFP (L).

(TIF)

**Author Contributions**

**Conceptualization:** Yanina-Yasmin Pesch, Guy Tanentzapf.

**Data curation:** Yanina-Yasmin Pesch.

**Formal analysis:** Yanina-Yasmin Pesch, Vivien Dang, Fayeza Islam, Priya Kaur, Ciaran R. McFarlane.

**Funding acquisition:** Filip Van Petegem, Guy Tanentzapf.

**Investigation:** Yanina-Yasmin Pesch, Vivien Dang, Fayeza Islam, Priya Kaur, Christopher M. Smendziuk, Ciaran R. McFarlane, Pierre-Yves Musso.

**Methodology:** Michael John Fairchild, Darius Camp, Christopher M. Smendziuk, Anat Messenberg.

**Project administration:** Guy Tanentzapf.

**Resources:** Michael John Fairchild, Darius Camp, Christopher M. Smendziuk, Anat Messenberg.

**Software:** Rosalyn Carr.

**Supervision:** Filip Van Petegem, Guy Tanentzapf.

**Validation:** Yanina-Yasmin Pesch.
**Visualization:** Rosalyn Carr, Ciaran R. McFarlane.

**Writing – original draft:** Yanina-Yasmin Pesch, Guy Tanentzapf.

**Writing – review & editing:** Yanina-Yasmin Pesch, Guy Tanentzapf.

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**Visualization:** Rosalyn Carr, Ciaran R. McFarlane.

**Writing – original draft:** Yanina-Yasmin Pesch, Guy Tanentzapf.

**Writing – review & editing:** Yanina-Yasmin Pesch, Guy Tanentzapf.

## References

1. Boyle M, Stephen D. n.d. 'Specification, Migration and Assembly of the Somatic Cells of the Drosophila Gonad', 11. https://doi.org/10.1242/dev.121.6.1815 PMID: 7600996

2. Kiger A. 'Stem Cell Self-Renewal Specified by JAK-STAT Activation in Response to a Support Cell Cue'. Science. 2001; 294 (5551): 2542-45. https://doi.org/10.1126/science.1066707 PMID: 11752574

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4. Hudry B, Goeij E, Mineo A, Gaspar P, Hadjjeconomou D, Studd C, et al. 'Sex Differences in Intestinal Carbohydrate Metabolism Promote Food Intake and Sperm Maturation'. Cell. 2019; 178 (4): 901-918. e16. https://doi.org/10.1016/j.cell.2019.07.029 PMID: 31398343

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