Regulation of Gonad Morphogenesis in Drosophila melanogaster by BTB Family Transcription Factors

During embryogenesis, primordial germ cells (PGCs) and somatic gonadal precursor cells (SGPs) migrate and coalesce to form the early gonad. A failure of the PGCs and SGPs to form a gonad with the proper architecture not only affects germ cell development, but can also lead to infertility. Therefore, it is critical to identify the molecular mechanisms that function within both the PGCs and SGPs to promote gonad morphogenesis. We have characterized the phenotypes of two genes, longitudinals lacking (lola) and ribbon (rib), that are required for the coalescence and compaction of the embryonic gonad in Drosophila melanogaster. rib and lola are expressed in the SGPs of the developing gonad, and genetic interaction analysis suggests these proteins cooperate to regulate gonad development. Both genes encode proteins with DNA binding motifs and a conserved protein-protein interaction domain, known as the Broad complex, Tramtrack, Bric-à-brac (BTB) domain. Through molecular modeling and yeast-two hybrid studies, we demonstrate that Rib and Lola homo- and heterodimerize via their BTB domains. In addition, analysis of the colocalization of Rib and Lola with marks of transcriptional activation and repression on polytene chromosomes reveals that Rib and Lola colocalize with both repressive and activating marks and with each other. While previous studies have identified Rib and Lola targets in other tissues, we find that Rib and Lola are likely to function via different downstream targets in the gonad. These results suggest that Rib and Lola act as dual-function transcription factors to cooperatively regulate embryonic gonad morphogenesis

Mesoderm develops normally in <i>rib</i> and <i>lola</i> mutants.

<p>(A) Oregon-R wild-type (WT) control (n = 10), (B) <i>lola</i>^{<i>46</i>.<i>38/22</i>.<i>05</i>} (n = 9), <i>and</i> (C) <i>rib</i>^{<i>35</i>.<i>14/55</i>.<i>25</i>} (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.</p

Calculated Interactions Energies for BTB Dimers.

<p>Calculated Interactions Energies for BTB Dimers.</p

Rib and Lola physically interact via their BTB domains by yeast two-hybrid analysis.

<p>(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.</p

Colocalization of Rib and Lola with marks of transcriptional activation and repression.

<p>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”‘) <i>forkhead</i>-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”‘) <i>forkhead</i>-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”‘) <i>forkhead</i>-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.</p

Protein modeling of Rib and Lola BTB domain interactions predicts heterodimerization and homodimerization.

<p>(A) Lola (red)-Rib (blue) BTB domain heterodimer. (B) Lola-Lola BTB domain homodimer. (C) Rib-Rib BTB domain homodimer.</p

Lola and Rib do not regulate expression of each other.

<p>(A-B”) Lola expression in <i>rib</i> heterozygous and homozygous mutant stage 15 gonads, posterior to the right. Anti-Lola (green) and anti-Traffic jam (TJ) marks SGPs (red). (A-A”) <i>rib</i>^<i>+/-</i> gonad (<i>rib</i>^{<i>35</i>.<i>14/+</i>} or <i>rib</i>^{<i>55</i>.<i>25/+</i>}) (n = 16). (B-B”) <i>rib</i>^{<i>35</i>.<i>14/55</i>.<i>25</i>} gonad (n = 11). (C-D”) Rib expression in <i>lola</i> heterozygous and homozygous mutant stage 15 gonads, posterior to the right. Anti-Rib (green) and anti-TJ marks SGPs (red). (C-C”) <i>lola</i>^<i>+/-</i> gonad (<i>lola</i>^{<i>46</i>.<i>38/+</i>} or <i>lola</i>^{<i>22</i>.<i>05/+</i>}) (n = 11). (D-D”) <i>lola</i>^{<i>46</i>.<i>38/22</i>.<i>05</i>} gonad (n = 11). The gonad is outlined with a dotted line. Scale bar: 10μm.</p

<i>rib and lola</i> genetically interact.

<p>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 <i>68-77-lacZ</i> 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: <i>Χ</i>² _{<i>22</i>, <i>0</i>.<i>05</i>} = 313.31, p<0.001.</p

Expression of Rib and Lola in the embryonic gonad.

<p>(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.</p