Fruitless Represses robo1 Transcription to Shape Male-Specific Neural Morphology and Behavior in Drosophila

Abstract

The Drosophila fruitless (fru) gene is regarded as a master regulator of the formation of male courtship circuitry, yet little is known about its molecular basis of action. We show that roundabout 1 (robo1) knockdown in females promotes formation of the male-specific neurite in sexually dimorphic mAL interneurons and that overexpression of the male-specific Fru(BM) diminishes the expression of Robo1 in the fly brain. Our electrophoretic mobility shift and reporter assays identify the 42-bp segment encompassing the palindrome sequence T T C G C T G C G C C G T G A A in the 5' UTR of robo1 exon1 as the Fru(BM)-responsive element. We find that ∼10-bp deletions in the palindrome sequence induce a loss of the male-specific neurite and disrupt male courtship patterns. This study paves the way for a thorough understanding of the mechanism whereby Fru proteins orchestrate transcription for the formation of courtship circuitry.

Molecular mechanisms underlying sexual differentiation of the nervous system

“…They suggest a model in which Fru M , acting via different isoforms, regulates sexually dimorphic expression of target genes that, individually, regulate different aspects of male courtship in a modular manner. This model is supported by a recent study from Yamamoto’s group in which they showed that the B isoform of Fru M represses robo1 in a specific cluster of neurons through a cis -regulatory sequence located in the promoter of robo1 [28•]. This results in a regulatory switch that guides these neurons down a male or female developmental pathway.…”

The mode of action of Fruitless: Is it an easy matter to switch the sex?

“…Bon was dispensable for FruM binding to the genomic sites, but HDAC1 and HP1a fail to bind to the FruM-positive sites in the absence of Bon, implying that FruM binds the site first, Bon binds the site second and then Bon recruits HDAC1 and/or HP1a. Ito et al 33 found that it is FruBM that removes Robo1 from male mAL neurons: FruBM binds to the robo1 promoter to repress its transcription. The likely scenario is that a FruM-containing complex switches chromatin states, thereby silencing or activating a large set of genes that are required in neurons for taking on the female or male fate ( Figure 2D).…”

“…The likely scenario is that a FruM-containing complex switches chromatin states, thereby silencing or activating a large set of genes that are required in neurons for taking on the female or male fate ( Figure 2D). 33 Pal1 contains the motif proposed as a FruM binding sequence by Neville et al 29 but does not contain any motif proposed by Dalton et al 35 Ito et al 33 discovered that a small deletion of a several base pair region in Pal1 of the robo1 promoter (eg, a 6 bp deletion occurring in the robo1 Δ1 allele) not only increased the number of male flies with no male-specific neurite but also impaired the male courtship posture, even in the flies carrying only one copy of the mutation, that is, heterozygotes. This result implies that Robo1 normally inhibits the ipsilateral neurite formation in females, whereas this Robo1-dependent inhibition is removed in males, allowing them to develop the ipsilateral neurite in mAL neurons ( Figure 3B).…”

“…48 Consistent with such synergy at the molecular level, overexpression of large Lola-Q mitigates cellular phenotypes of fru loss-of-function; e.g., it restores the male-specific neurite otherwise lost in fru mutant males ( Figure 4C). 33 Peptide sequencing by Edman degradation showed that small Lola-Q lacks N-terminal amino acid residues 1-263 of large Lola-Q, including the entire BTB domain. These observations suggest that female-specific small Lola-Q is indeed a feminizing factor, whereas large Lola-Q with male-biased expression is a masculinizing factor of fru-positive sexually dimorphic neurons.…”

“…Possible effects of these mutations on sex-specific neural development were examined by clonally labeling the mAL cluster of fru-positive neurons, because this particular cluster displays conspicuous sex differences: the number of cells composing the cluster is 5 in females and 30 in males;the ipsilateral neurite is present in some male neurons but totally absent in all female neurons; the tip of the contralateral neurite in the subesophageal ganglion always bifurcates in females whereas it has a comblike appearance without a bifurcation in males 7 (Figure 2D). However, potential FruM downstream genes have impact on a select aspect of the sexual dimorphism in mAL neurons (Figure 2E): loss of three major cell death-inducing genes, grim, hid and reaper, affects primarily the sex difference in the number of mAL neurons 7 ; the transcription factor Hunchback selectively acts to shape the sexually-dimorphic branching of the contralateral neurite 32 ; robo1 (see below) specifically affects the sexdependent presence or absence of the ipsilateral neurite 33. A single cell clone analysis unequivocally showed that reductions in fru or HDAC1 gene activity increased the female-type mAL neurons, whereas similar reductions in HP1a gene activity in fru hypomorphic males increased male-type mAL neurons.…”