Fig 1.
lola and rib mutants exhibit defects in gonad morphogenesis.
(A) Schematic of the stages of embryonic gonad formation. At stage 12 SGP (red) clusters and PGCs (green) begin to intermingle. At stage 13 SGP clusters fuse and coalesce with the PGCs, sending out extensions around PGCs to ensheath them. By the end of stage 15 SGPs and PGCs compact to form a spherical gonad. (B) Molecular structure of Lola and Rib with BTB domains (light blue), Lola common region (dark blue), zinc finger DNA binding motifs (ZF; yellow), pipsqueak DNA binding motif (PSQ; pink), and the corresponding mutations found in alleles used in this study. (C-G) Stage 15 mutant embryos immunostained for the 68-77-lacZ enhancer trap, which labels the cytoplasm of SGPs for analysis of gonad morphology. Posterior to the right. (C) Control embryos expressing the 68-77-lacZ enhancer trap. Scale bar: 10μm. (D-E) lola46.38/22.05 embryonic gonads exhibiting fusion (D) and compaction (E) defects. (E-F) rib35.14/55.25 embryonic gonads exhibiting (F) fusion and (G) compaction defects. (H) Quantification of the frequency of gonad phenotypes observed in control, lola mutant and rib mutant embryos. Gonad morphology was assessed using the 68-77-lacZ enhancer trap. The following phenotypes were scored: fusion (red), compaction (blue) and wild-type (green). A chi-square test was performed to test the null hypothesis that the phenotypic ratios will be the same across all genotypes. Results allow us to reject the null hypothesis: Χ2 28, 0.05 = 118.95, p<0.001.
Table 1.
Calculated Interactions Energies for BTB Dimers.
Fig 2.
Expression of Rib and Lola in the embryonic gonad.
(A-A”‘) Expression of Lola in an Oregon-R stage 13 gonad. (A) Anti-Lola (green). (A’) Anti-Traffic jam (TJ) marks somatic gonadal precursors (SGPs; red). (A”) Anti-Vasa marks primordial germ cells (PGCs; blue). (A”‘) Merged image with anti-Lola (green), anti-TJ (SGPs; red), and anti-Vasa (PGCs; blue). (B-B”‘) Expression of Lola in an Oregon-R stage 15 gonad. (B) Anti-Lola (green). (B’) Anti-TJ (SGPs; red). (B”) Anti-Vasa (PGCs; blue). (B”‘) Merged image with anti-Lola (green), anti-TJ (SGPs; red), and anti-Vasa (PGCs; blue). (C-C”‘) Expression of Rib in an Oregon-R stage 13 gonad. (C) Anti-Rib (green). (C’) Anti-TJ (SGPs; red). (C”) Anti-Vasa (PGCs; blue). (C”‘) Merged image with anti-Rib (green), anti-TJ (SGPs; red), anti-Vasa (PGCs; blue). Same scale as (A). (D-D”‘) Expression of Rib in an Oregon-R stage 15 gonad. Same scale as (B). (D) Anti-Rib (green). (D’) Anti-TJ (SGPs; red). (D”) Anti-Vasa (PGCs; blue). (D”‘) Merged image with anti-Rib (green), anti-TJ (SGPs; red), anti-Vasa (PGCs; blue). (E-E”‘) Colocalization of Rib and Lola in an Oregon-R stage 13 gonad. Same scale as (A). (E) Anti-Rib (green). (E’) Anti-Lola (red). (E”) Anti-TJ (SGPs; blue). (E”‘) Merge of anti-Rib (green) and anti-Lola (red). (F-F”‘) Colocalization of Rib and Lola in an Oregon-R stage 15 gonad. Same scale as (B). (F) Anti-Rib (green). (F’) Anti-Lola (red). (F”) Anti-TJ (SGPs; blue). (F”‘) Merge of anti-Rib (green) and anti-Lola (red). Gonads are outlined by dotted lines. Areas of high Rib-Lola colocalization are indicated by arrows. For all images posterior is to the right. Scale bars: 10μm.
Fig 3.
Mesoderm develops normally in rib and lola mutants.
(A) Oregon-R wild-type (WT) control (n = 10), (B) lola46.38/22.05 (n = 9), and (C) rib35.14/55.25 (n = 5) stage 12 embryos immunostained for the visceral mesodermal marker Fasciclin 3. For all images posterior is to the right. Scale bar: 50 μm.
Fig 4.
rib and lola genetically interact.
Graph of phenotypic frequency for stage 15 embryonic gonads. The following gonad phenotypes were scored: fusion (red), compaction (blue) and wild-type (green). Gonads were scored by staining somatic gonadal precursor cells for the 68-77-lacZ enhancer trap. A chi-square test was performed to test the null hypothesis that the phenotype ratios would be the same across all genotypes. Results allow us to reject the null hypothesis: Χ2 22, 0.05 = 313.31, p<0.001.
Fig 5.
Lola and Rib do not regulate expression of each other.
(A-B”) Lola expression in rib heterozygous and homozygous mutant stage 15 gonads, posterior to the right. Anti-Lola (green) and anti-Traffic jam (TJ) marks SGPs (red). (A-A”) rib+/- gonad (rib35.14/+ or rib55.25/+) (n = 16). (B-B”) rib35.14/55.25 gonad (n = 11). (C-D”) Rib expression in lola heterozygous and homozygous mutant stage 15 gonads, posterior to the right. Anti-Rib (green) and anti-TJ marks SGPs (red). (C-C”) lola+/- gonad (lola46.38/+ or lola22.05/+) (n = 11). (D-D”) lola46.38/22.05 gonad (n = 11). The gonad is outlined with a dotted line. Scale bar: 10μm.
Fig 6.
Protein modeling of Rib and Lola BTB domain interactions predicts heterodimerization and homodimerization.
(A) Lola (red)-Rib (blue) BTB domain heterodimer. (B) Lola-Lola BTB domain homodimer. (C) Rib-Rib BTB domain homodimer.
Fig 7.
Rib and Lola physically interact via their BTB domains by yeast two-hybrid analysis.
(A-A’) Growth on SD-Leu-Trp plates illustrates successful yeast mating. (B-B’) SD-Leu-Trp-His-Ade plates with X-α-gal and Aureobasidin A were used to test for interaction of activation domain (AD) and DNA binding domain (BD) fusion proteins.
Fig 8.
Colocalization of Rib and Lola with marks of transcriptional activation and repression.
Immunofluorescence staining of polytene chromosomes of third instar larval salivary gland. (A-A”‘) Oregon-R polytene chromosomes stained with anti-Lola (green) and anti-H3K27me3 (red), with the merge showing areas of colocalization. (A”‘) Zoomed images of (A-A”). (B-B”‘) Oregon-R polytene chromosomes stained with anti-Lola (green) and anti-PolIIser5 (red), with the merge showing areas of colocalization. (B”‘) Zoomed images of (B-B”). (C-C”‘) forkhead-Gal4; UAS-3xHA-Rib polytene chromosomes stained with anti-HA (Rib; green) and anti-H3K27me3 (red), with the merge showing areas of colocalization. (C”‘) Zoomed images of (C-C”). (D-D”‘) forkhead-Gal4; UAS-3xHA-Rib polytene chromosomes stained with anti-HA (Rib; green) and anti-PolIIser5 (red), with the merge showing areas of colocalization. (D”‘) Zoomed images of (D-D”). (E-E”‘) forkhead-Gal4; UAS-3xHA-Rib polytene chromosomes stained with anti-Lola (green) and anti-HA (Rib; red), with the merge showing areas of colocalization. (E”‘) Zoomed images of (E-E”). Arrowheads indicate region with Rib lacking Lola. Scale bars in unzoomed images: 10μm. Scale bars in zoomed images: 2μm. Arrows indicate colocalization. For each experiment a minimum of 10 polytene chromosomes were examined.