Figure 1.
Schematic drawing of gonad formation in wild type and mutants without PGCs.
(Top and middle panels) Gonad coalescence in wild type. SGPs (blue) are specified as three clusters in parasegments (PSs) 10–12. At stages 11–12, PGCs (red) migrate to associate with somatic gonads. At stage 13, SGPs and PGCs align in the future gonad region. At stage 14, SGPs and PGCs undergo compaction to form the embryonic gonad. (Bottom panels) Normal gonad coalescence in mutants without PGCs. In the absence of PGCs, SGPs are specified normally and are able to complete gonad coalescence.
Figure 2.
The enaC14-06 allele affects gonad coalescence.
(A–E) SGPs were detected by in situ hybridization with a 412 retrotransposon RNA probe (blue). PGCs were labeled with anti-Vasa (Vas) antibody (brown). The brown signal in a segmental pattern is β-galastosidase from the balancer chromosome. In wild type, individual SGP clusters align with PGCs at stage 13 (A) and undergo coalescence to form tightly associated embryonic gonads at stage 14 to 16 (B). In enaC14-06 mutants, individual SGP clusters fail or delay to adhere at stage 13, remaining instead in three clusters (arrows in C). Subsequently, mutant gonads fail to complete coalescence resulting in elongated (bracket in D) or split gonads (arrows in E). (F–H) Ena protein detected with anti-Ena antibody. Ena protein is expressed ubiquitously in the wild type (F). Ena protein was not detected in enaC14-06 mutants either by tissue-immuno fluorescence (G) or Western blotting (H). Scale bar in (A) represents 50 µm.
Figure 3.
Ena is expressed in the SGPs during gonad coalescence.
(A–C) Stage-12 embryo stained with the anti-Ena antibody (magenta in A–B, white in C) and anti-Vas antibody to detect PGCs (green). The posterior half of the embryo is shown in (A), migrating PGCs are magnified in (B, C) (asterisks). Ena protein is found at the cell membranes (A) but not in migrating PGCs (B, C). (D–L) Ena expression during gonad coalescence. Wild-type embryos were stained with anti-Ena (magenta), anti-Zfh-1 antibodies, which mark the nuclei of the fat body and SGPs (green), and anti-Vas (PGCs in blue). Ena levels in the SGPs were indistinguishable from those in the surrounding tissues (D, high magnification in E, F). Ena expression in the SGPs was more pronounced in the mature gonad (G, J, high magnification in H–I and K–L). The scale bars in (A and D), (B), and (E) represent 50 µm, 10 µm, and 20 µm, respectively.
Figure 4.
ena functions in the soma for proper gonad coalescence.
(A–F) Rescue experiment of ena mutants. The coalescence phenotype at stages 15–16 was categorized into severe (green), intermediate (red), and normal classes (blue) as illustrated in (A), and number of gonads scored is indicated above the graph (B). Germline expression of Ena by nanos (nos)-Gal4 did not show any rescue activity (B) albeit the Ena protein derived from the transgene was detected in PGCs (blue in E and F). Ena expression in the mesoderm by 24B-Gal4 partially rescued the compaction phenotype in the mutants (B). (G–J) ena function was tested in a maternal oskar mutant background lacking PGCs. The 412 retrotransposon probe was used to label SGPs (blue). In maternal oskar mutants, SGPs were specified normally and compacted into spherical ‘gonads’ (G and H). In ena (zygotic), oskar (maternal) double mutants, SGP clusters failed to adhere at stage 13 (I), resulting in elongated gonads similar to those in zygotic ena mutants (J). (K–L) The stage-15 embryos were stained with anti-Serpent and anti-Vas antibodies to detect the fat body (magenta, bracket) and PGCs (green). The fat body appears unaffected by the ena mutation. Scale bars in (A) and (D) represent 50 µm.
Figure 5.
Live imaging of the SGPs in wild type and ena mutants.
SGPs labeled with PD-six4-egfp::moesin were imaged in wild-type and ena mutant embryos. (A–I) Still images from Movies S1 (A–C) and S2 (D–I) in wild type. At early stage 13, some SGPs had broad migratory protrusions (arrowheads in A), which were lost as gonad coalescence proceeded, and SGPs in the anterior and posterior regions moved to the middle of the gonad (B–C). During coalescence, the SGPs extended protrusions between PGCs (A–I). These protrusions were highly dynamic, and elongated, branched, and connected with each other to encapsulate the PGCs (arrows in D–I). (J–O) Still images from Movie S3 in ena mutants. The ena mutant gonad imaged in (J–O) displayed moderate phenotype similar to the gonad shown in Figure 6C and D. The mutant SGPs normally ensheathed PGCs; however, the anterior and posterior SGPs were unable to move inward resulting in incomplete coalescence. The scale bar in (A) represents 10 µm.
Figure 6.
ena is dispensable for the formation of cytoplasmic protrusions in SGPs.
(A–F) Wild-type and ena mutant embryos with the Psix4-egfp::moesin transgene were double stained with anti-GFP (green) and anti-Vas (magenta) antibodies. Labeled gonads at stages 15–16 were scanned and rendered into 3D images. Z projection (A, C, E) and selected single frames (B, D, F) are presented. In the wild-type gonad, SGPs extended numerous protrusions to ensheath PGCs (A, arrowheads in B). In ena mutants, SGPs appeared to have normal cytoplasmic extensions surrounding the PGCs (C, E, arrowheads in D, F). Some mutant SGPs, mostly in the anterior region, severely extend (arrows in E), resulting in an irregularly shaped gonad.
Figure 7.
ena is required for rearrangement and cell shape changes of SGPs.
(A) The circularity of individual SGPs was calculated in the anterior, middle, and posterior regions of the gonad. Circularity (see Materials and methods) was similar between the wild type and mutants at stage 13. However, at stages 15–16, circularity increased with compaction in wild-type SGPs. In ena mutants, SGP circularity was unchanged in the later stages, indicating that mutant SGPs remain extended (*P<0.0001). The number of cells examined is shown in the bar. (B) SGP orientation relative to the anterior-posterior (AP) axis of the gonad was measured. At stage 13, wild-type and ena mutant SGPs were oriented along the AP axis of the gonad. At stages 15–16, the long axis of wild- type SGPs were oriented away from the AP axis; however, ena mutant SGPs remained aligned with the AP axis. The number of cells examined is shown in the middle of the graph. Error bars in (A) represent standard error.
Figure 8.
ena shows moderate genetic interaction with DE-cad in gonad compaction.
To test for genetic interactions between ena and DE-cad (shg), the gonad coalescence phenotype was examined in transheterozygotes of ena with shg mutant alleles. The coalescence phenotype at stages 15–16 was categorized into severe (green), intermediate (red), and normal classes (blue). ena and shg showed weak genetic interaction in gonad compaction. The number of gonads scored is indicated above the graph.
Figure 9.
The ena mutation alters intracellular localization of DE-cad in SGPs.
(A, B) The DE-cad protein in control and ena mutant embryos was detected by Western Blot (A) and the expression level of DE-cad normalized against α-tubulin expression was plotted in the graph (B). (C–H) Wild-type and ena mutant SGPs were stained with the DE-cad antibody, and the signal intensity on the outer membrane (red) or SGP-SGP boundary (blue) was measured (middle and left panels). The ratio of the signal intensity on the outer membrane compared to that on the SGP-SGP boundary was calculated at stage 13 and stages 15–16 (middle panel). In wild type, DE-cad becomes enriched at the SGP-SGP boundary as gonad compaction proceeds (**P<0.01) (D–F). This process was affected in ena mutants (F–H). (I–K) Wild-type SGPs were stained with the anti-Ena antibody (I, J), and the ratio of the signal intensity on the outer membrane compared to that on the SGP-SGP boundary was plotted at stage 13 and stages 15–16 (K). Ena localization shifts towards the SGP-SGP boundary at stages 15–16 (*P<0.05). The number of outer membranes versus SGP-SGP boundaries scored is indicated above the graph (F, K). Error bars in (B, F, and K) represent standard error.
Figure 10.
Mathematical simulation of gonad compaction.
(A) A scheme of the Cellular Potts model (CPM). In the CPM, each cell is described over a region of multiple sites on a square lattice, and the shape of individual cells is determined by simulating the minimization process of a given potential energy for the system (see Materials and Methods for details). Briefly, in each Monte Carlo step (MCS), a flip of a randomly chosen pixel at the cell boundary (see the ij-th pixel) is adopted with the probability determined by the difference in potential energy between the two configurations. (B) Left: The average values of gonad compaction (circularity) between 12,000 and 20,000 MCS were calculated, and are shown in a color map against different values of ESGP-SGP and ESGP-out. The circularity increases for smaller contracting forces (larger adhesive forces) among SGPs, and for larger contracting force along the surfaces between SGPs and the outer space. Right: The cell configurations obtained from the CPM at 19,660 MCS. Values of ESGP-SGP and ESGP-out are indicated with the corresponding number in the left panel. Other parameter values are described in Methods. (C–D) Temporal changes in gonad morphology in the CPM. Selected frames from Movies S4 (C) and S5 (D) are shown. (C) and (D) correspond to 1 and 2 in the phase diagram in (B). (E) A mechanical view of gonad compaction. For clarity, only SGPs are shown. Blue lines indicate the SGP-SGP contact surface. Redistribution of DE-cad to the SGP-SGP surface generates a large adhesive force between the SGPs (i.e., small ESGP-SGP), which increases the SGP-SGP contact surface. The adhesive force, therefore, functions as a glue to move the SGPs inward (left).