Little branch, big difference

For many of us, middle school health class provided the basic biological and anatomical differences between males and females. While we can recognize physical differences from a young age and learn about the differing reproductive organs in our pre-teen years, many sex-specific characteristics and behaviors are more difficult to understand; particularly those in the brain. What causes structural and functional differences in the brains of males versus females?

In a 2016 publication in Current Biology, Dr. Hiroki Ito, Dr. Daisuke Yamamoto and their team at Tohoku University in Sendai, Japan dove deeper into one of these sex differences and its effect on the courtship behaviors in flies, Drosophila melanogaster. By studying and manipulating the structure of a certain group of sexually dimorphic neurons called the mAL neurons (which function to inhibit male courtship), the researchers were able to discover the genes and mechanisms that influence their dimorphic development.

The mAL neurons differ in males and females in three ways. First, male mAL neurons consist of 30 cells while females have only 5 cells. Second, male mAL neurons project a neurite on the contralateral and ipsilateral side of the brain while females have only the contralateral projection. (Contralateral refers to having projections on the opposite side while ipsilateral refers to the same side.) Third, female mAL neurons have forked arborization of the contralateral projection while males have a horsetail-like structure at the end of their contralateral neurite. In this study, the researchers focused on why it is that the ipsilateral neurite develops in males but not in females.

Dr. Ito and his colleagues found that the male-specific transcription factor known as fruitless (FruM ) binds to a sequence in the promoter region of a gene called roundabout 1 (robo1) whose normal function is to block the formation of the mAL’s male-specific, ipsilateral projection. When FruM binds to robo1’s promoter region, it represses robo1 expression in the male. In females, FruM is not expressed, which thereby disinhibits robo1 expression in females and the ipsilateral branch fails to form.

How did the researchers figure this out? First, the team performed an experiment in which they knocked down robo1 function specifically in developing mAL neurons of females. In robo1-deficient females, mAL neurons developed an ipsilateral projection. However, when the same procedure was done in males, there was no difference in projections between wild-type and robo1-deficient males (Figure 1c–f). These findings suggested to the researchers that the formation of the ipsilateral neurite is normally inhibited in females by the function of robo1.

Next, Dr. Ito and his colleagues determined the connection between FruM and robo1 in regulating the formation of the ipsilateral neurite in male Drosophila. They found that male mAL neurons without functional FruM lost the ipsilateral projection, as if robo1 expression was gained in the mutant males. Similarly, female flies that had overexpressed FruM also did not possess ipsilateral neurites. These findings led the authors to propose a genetic pathway in which FruM in males represses robo1 which in turn represses the development of the mAL ipsilateral neurite (figure 2).

How might FruM repress robo1 expression in males? Since FruM is a transcription factor, the most obvious hypothesis would be that FruM directly represses the transcription of robo1 in males; in females, this repression would be relieved due to the absence of FruM, and robo1 would then be permitted to be expressed.

To test this idea, the team went on to see if FruM directly binds sequences near the robo1 promoter. Dr. Ito and his colleagues cloned a series of DNA fragments of various sizes surrounding the robo1 transcriptional start. Each fragment was then hooked up to the luciferase gene (luciferase is the protein that makes fireflies light up), and the DNA constructs were introduced into cells. Most of the fragments drove some luciferase expression. Interestingly, when they simultaneously introduced FruM protein into the cells, the level of luciferase activity dropped dramatically, indicating that FruM protein has the ability to repress transcription. Eventually they were able to identify a 42-bp sequence, called FROS (FruM Response Obligatory Sequence), to which FruM protein physically binds. This sequence is located very close to the promoter of robo1. Moreover, Dr. Ito and his colleagues were able to generate mutant flies in which the FROS was deleted. Remarkably, the mAL neurons of flies lacking this binding sequence did not develop a normal, full size ipsilateral projection.

Focusing on the mAL neurons, Dr. Hiroki Ito and his colleagues were able to discover the mechanisms that led to the development of a sexual diorphism in neural anatomy. They were able to show that the male isoform of the transcription factor, FruM, represses the gene robo1 in male flies, which allows for the formation of the male-specific ipsilateral neurite. Being that females do not express any version of FruM, robo1 expression is not repressed and ipsilateral neurite formation is restricted. These findings prompt new questions and potential studies for the team: Does robo1 have multiple roles in regulating mAL neural anatomy? Can the binding site be narrowed down even more? And what else is the FROS sequence responsible for? An immense amount of research is still required to understand the molecular bases of mAL dimorphisms in Drosophila, but Dr. Ito and his team have made huge strides in the field and have paved the way for many future studies.