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teiresias: Biological Overview | References

This study aims at identifying transcriptional targets of FruitlessBM (FruBM), which represents the major isoform of male-specific FruM transcription factors that induce neural sexual dimorphisms. A promoter of the axon-guidance factor gene robo1 carries the 16-bp palindrome motif Pal1, to which FruM binds. A genome-wide search for Pal1-homologous sequences yielded ~200 candidate genes. Among these, CG17716 potentially encodes a transmembrane protein with extracellular immunoglobulin (Ig)-like domains similar to Robo1. Indeed, FruBM overexpression reduced CG17716 mRNA and protein expression. In the fru-expressing mAL neuron cluster exhibiting sexual dimorphism, it was found that CG17716 knockdown in female neurons completely transformed all neurites to the male-type. Conversely, CG17716 overexpression suppressed male-specific midline crossing of fru-expressing sensory axons. CG17716 was renamed teiresias (tei) based on this feminizing function. It is hypothesized that Tei interacts with other Ig superfamily transmembrane proteins, including Robo1, to feminize the neurite patterns in females, whereas FruBM represses tei transcription in males (Sato, 2020).

Reproductive success in many male animals relies on their behavioral performance during mating attempts with a female1. The neural circuitry that controls mating behavior has thus evolved under the strong pressure of sexual selection. Such an evolutionary drive led to the development of sexual dimorphisms in the neural circuitry and neurons that compose this circuitry1. Drosophila melanogaster is an outstanding model organism highly amenable to genetic dissection of complex traits, including mating behavior2. In this organism, the transcription factor gene fruitless (fru) plays a key role in organizing the sexually dimorphic circuitry for mating behavior by specifying sex-specific neuronal structures during development (Sato, 2020).

Among the four promoters identified in the fru gene, the most distal one (the P1 promoter) is dedicated to sex-specific functions ascribed to the male-specific translation of P1-derived mRNAs. As a result of sex-specific splicing of the fru-P1 primary transcript, only male mRNAs have a long ORF encoding male-specific functional fru proteins, called FruM. These proteins are translated as five isoforms, FruAM, FruBM, FruCM, FruDM, and FruEM, which share a common BTB domain in their N-terminus, but which each have a unique C-terminus. The most prevalent isoform is FruBM, which has two zinc-finger motifs in the C-terminus, and forms a complex with chromatin regulators such as the TIF1 homolog Bonus (Bon), histone deacetylase 1 (HDAC1), and heterochromatin protein 1a (HP1a), to bind to more than 100 target sites on the genome for transcriptional regulation of downstream genes. The best-characterized FruBM target is robo1, which encodes a transmembrane protein belonging to the immunoglobulin superfamily with an axon-guidance role in the developing nervous system. FruBM binds to a 42-bp promoter segment of the robo1 gene named the FruBM Response Obligatory Sequence (FROS), which contains a characteristic palindrome sequence of 16 nucleotides (Pal1). Partial deletion of Pal1 in the robo1 gene impairs neural sexual differentiation and mating behavior in male flies, indicating that Pal1 is pivotal for FruBM in order to execute its sexual functions (Sato, 2020).

Many of the fru-expressing neurons are sexually dimorphic, including neurons composing the mAL cluster. The mAL cluster displays sexual dimorphisms in the following four respects: (i) the number of neurons in the cluster (~5 in females vs. ~30 in males); (ii) the absence (females) or presence (males) of a neuronal subset carrying the ipsilateral neurite; (iii) the branched (females) vs. unbranched (males) tip structure of the descending contralateral neurite; and (iv) the focal (females) or expanded (males) terminal arbor distribution of the ascending contralateral neurite. While these four types of sexual dimorphisms of mAL neurons are established in a fru-dependent manner, they are also regulated separately and independently from each other. For example, Hunchback knockdown in male mAL neurons feminizes only the descending contralateral neurite, whereas robo1 knockdown feminizes only the ipsilateral neurite. On the other hand, loss of the cell-death genes, reaper, grim and head involution defective, increases the number of cells composing the mAL cluster from ~5 to ~30 in females, but has no effect in males. Therefore, FruBM likely interacts with different cofactors for transcription regulation, and/or FruBM acts on different targets to establish each of the four sexually dimorphic features of mAL neurons. With the aim of obtaining additional FruBM transcriptional targets for the establishment of neural sexual dimorphisms, this study examined the effects of knocking down the genes harboring a Pal1-homologous motif on mAL neuron structures. teiresias (tei), encoding another immunoglobulin-superfamily member with a transmembrane domain, was identified. Remarkably, knockdown of tei in female mAL neurons completely masculinized all three neurite characteristics, leaving the cell number unaffected. tei knockdown also masculinized fru-expressing sensory neurons and mcALa interneurons in females. It is proposed that the Tei protein functions as a common receptor and interacts with a second receptor, e.g., robo1, which confers the ligand specificity on the heteromeric receptor complex, producing distinct neuronal populations that are diversified in their sex-specific structures (Sato, 2020).

tei, a new transcriptional target of FruBM, was identified based on the in silico search for genes harboring the FruBM-binding consensus sequence and subsequent analysis of gene knockdown effects on sexually dimorphic neurite structures. Tei is a putative transmembrane protein carrying multiple immunoglobulin-like repeats in the extracellular portion. This structure is very much alike that of Robo1, the product of the best-characterized FruBM target gene. Indeed, tei knockdown in female mAL neurons induced the male-specific ipsilateral neurite as did robo1 knockdown, revealing a functional similarity between tei and robo1 in terms of the feminizing effect on this particular structure. Nonetheless, tei was distinctly different from robo1 in that tei is also required for the female-typical shaping of ascending as well as descending contralateral neurites, in which robo1 has no role. Robo1 has been shown to operate as a receptor for the secreted ligand Slit, activating several signal transduction pathways depending on the developmental context in both invertebrates and vertebrates. Promiscuous interactions of Robo1 with other transmembrane receptors in cis have been implicated as a basis for the versatile performances of Robo1 in multiple developmental contexts. The lack of the cytoplasmic portion in Tei might suggest that Tei contributes to the ligand specificity whereas the specificity of intracellular signal transduction following heteromeric receptor activation is determined by Robo1 or another Tei partner that carries the C-terminal cytoplasmic domain. It is therefore plausible that Tei is one of these transmembrane receptors that cooperate with Robo1, and likely initiates together an intracellular biochemical cascade which ultimately inhibits the male-specific ipsilateral neurite from forming in an mAL neuron. Perhaps Tei associates with transmembrane receptors other than Robo1 in specifying the sex-specific structure in the ascending and descending contralateral neurites of an mAL neuron. Systematic knockdown in mAL neurons of genes encoding putative transmembrane receptors with immunoglobulin-like domains other than Robo protein members might lead to the identification of additional Tei partner proteins that are involved in the sex-type specification of the contralateral neurites (Sato, 2020).

Despite its striking effects on neurite structures, tei knockdown had no effect on the sexually dimorphism in the number of mAL neurons. This contrasts with the effects of fru loss-of-function mutation, which reduces the number of cells composing the mAL cluster in males from ~30 to ~5, the cell number typical of the mAL cluster in a wild-type female. Removal of three major cell-death genes, hid, grim and rpr, in female mAL neurons increased the number of cells composing the female mAL cluster up to 29 (the mean cell number was 19). This observation led to the proposition that cell death is the primary cause of the smaller number of cells composing the mAL cluster in females. Moreover, the sex difference in the cell number of fru-expressing neurons has recently been attributed to a difference in proliferation in addition to a difference in cell death: male neuroblasts produce more cells than female neuroblasts, so that males have a larger number of cells even after blocking cell death. These considerations lead to the supposition that FruM proteins separately control the neurite sex-type and the cell number sex-type. In this study it was noted that the Pal1-homologous motif was not found in or around any of the hid, grim, and rpr loci, suggesting they are not direct transcriptional targets of FruM proteins. Alternatively, transcription of these genes might be regulated by a FruM isoform other than FruBM. In fact, the mAL cell number was shown to be affected by the fruB2 (null for FruEM) as well as the fruC1 (null for FruBM) mutation (Sato, 2020).

The 16-bp Pal1 core motif is located within the 42-bp FruBM-binding site, FROS. This motif was first identified in the robo1 gene by a combination of reporter assays, electromobility-shift assays, and CRISPR-Cas9-mediated in vivo mutagenesis in conjunction with phenotypic characterizations of single-cell clones of mAL neurons and mutant-fly behavior. Using different strategies, two other research groups determined the binding consensus sequences for FruAM and FruEM in addition to FruBM. Neville (2014) used the DNA adenosine methyltransferase identification (DamID) method, in which a Fru-fused bacterial methylase methylates the DNA around the genomic region to which the fru moiety of the fusion protein binds. One of the reported FruBM-binding motifs in the DamID-based search revealed a 4/6 match when compared with Pal1, yet robo1 was not obtained as a putative FruBM target in that study. Dalton (2013) used in vitro screening of oligo-DNAs with random sequences for binding to fru zinc-finger motifs fused to glutathione S-transferase, revealing a binding consensus sequence for each isoform, but none of these had a similarity to Pal1. Of note, CG17716 (tei) has been included, together with another ~1400 genes, in a list of potential FruBM targets having a deduced FruBM-binding sequence by Neville (2014). Reliable determination of consensus sequences for the binding of fru isoforms and rigorous identification of fru transcriptional targets are indispensable for obtaining a more complete picture of the molecular mechanisms underlying sex-specific circuit formation (Sato, 2020).

Search PubMed for articles about Drosophila Teiresias

Brown, K. E., Keller, P. J., Ramialison, M., Rembold, M., Stelzer, E. H., Loosli, F. and Wittbrodt, J. (2010). Nlcam modulates midline convergence during anterior neural plate morphogenesis. Dev Biol 339(1): 14-25. PubMed ID: 20005219

Dalton, J. E., Fear, J. M., Knott, S., Baker, B. S., McIntyre, L. M. and Arbeitman, M. N. (2013). Male-specific Fruitless isoforms have different regulatory roles conferred by distinct zinc finger DNA binding domains. BMC Genomics 14: 659. PubMed ID: 24074028

Neville, M. C., Nojima, T., Ashley, E., Parker, D. J., Walker, J., Southall, T., Van de Sande, B., Marques, A. C., Fischer, B., Brand, A. H., Russell, S., Ritchie, M. G., Aerts, S. and Goodwin, S. F. (2014). Male-specific fruitless isoforms target neurodevelopmental genes to specify a sexually dimorphic nervous system. Curr Biol 24: 229-241. PubMed ID: 24440396

Sato, K., Ito, H. and Yamamoto, D. (2020). teiresias, a Fruitless target gene encoding an immunoglobulin-superfamily transmembrane protein, is required for neuronal feminization in Drosophila. Commun Biol 3(1): 598. PubMed ID: 33087851

date revised: 25 May 2021

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