Fig 5. Mutations in the Zpg D50 channel pore residue result in germ cell differentiation defects.

(A) Homology model of Zpg showing the position of aspartate 50 (D50) within the channel pore. (A’) D50 is predicted to form a hydrogen bond with glutamine 49 (Q49) of the adjacent Zpg subunit. (A”’) Simplified model highlighting the location of D50 (marked in pink) in the first extracellular loop. (B-F) Colocalization of wildtype endogenous Zpg and GFP-tagged, mutated Zpg. Flies heterozygous for a null allele of zpg but also containing one copy of the wildtype genomic zpg rescue construct (zpg GFP::GR; B-B’), no rescue construct (C-C’), one copy of the zpg D50A mutant rescue construct (D-D’), one copy of the zpg D50R mutant rescue construct (E-E’) and one copy of the zpg D50K mutant rescue construct (F-F’), the GFP-tagged D50 mutants show strong colocalization with the endogenous Zpg at the membrane. This high degree of colocalization is also revealed by the quantification of the Pearson coefficient between the GFP and Zpg antibody staining (G). (H-Q) Staining for the mitotic germ cell marker Vasa and the late-stage germ cell marker Boule. In the wildtype, Vasa staining is mostly concentrated in the apical part of the testis (H), whereas Boule marks meiotic cysts and long, parallel bundles of spermatids (I). In zpg null mutant testes little Vasa (J) and no Boule (K) signal is detected. In both zpg D50A (L) and zpg D50R (N) testes, the Vasa signal is strong and broadly localized. However, in zpg D50A testes, Vasa-positive cysts are abnormally found throughout the entire testis (L), and defective cysts can be observed in both mutants (circled in L, arrowhead in N). In addition, Boule staining in testes of zpg D50A (M) and zpg D50R (O) mutants reveals disorganized spermatid bundles. While zpg D50K mutant testes have a larger number of germ cells and larger mitotic cysts compared to zpg null mutants (P), they fail to reach meiosis (Q). Quantification of (R) germline stem cells (GSCs), (S) Zfh-1-positive cells, (T) Tj-positive cells, (U) Eya-positive cells, (V) spermatid bundles, and (W) fertility data. The data indicates late germ cell differentiation defects in zpg D50A and zpg D50R mutants and a stronger phenotype closer to the null mutant in zpg D50K mutants. Hubs are either encircled or indicated by asterisks. Scale bars represent 50 μm, as indicated above them. n>30 single crosses per genotype for fertility tests. p-values are for difference from wildtype and indicated by asterisks with *p<0.05, **p<0.01, ***p<0.001.