Lee, KyungHwa et al. (2009) International Worm Meeting "The DM gene mab-3 generates a functional sex difference in the AWA neurons."

Lee, KyungHwa, Portman, Douglas

[International Worm Meeting, 2009]

To understand how the "sex" of the nervous system influences its function, we are studying sex differences in olfaction. Previously, we have observed significant sex differences in responses to single odorants and in an "olfactory preference" assay. These sex differences do not require gonadal signaling or the male-specific CEM sensory neurons. Instead, they arise from intrinsic sex differences in shared or "core" neurons. To directly address the role of neural sex, we manipulated sex determination genes in the nervous system. Interestingly, we have found that masculinization of the hermaphrodite nervous system through the pan-neural overexpression of the sex-determination gene fem-3 is sufficient to generate male-like olfactory preference. Conversely, expression of activated tra-2 in the male nervous system gives rise to hermaphrodite-like olfactory preference. To identify the neuronal foci that underlie these sex differences in behavior, we specifically sex-reversed the AWA olfactory neurons and examined behavioral responses in the diacetyl-pyrazine (da-py) preference assay. In wild-type animals, hermaphrodites tend to prefer da, while males prefer py. In contrast, sex-reversing AWA sex-reverses the behavioral phenotypes of both males and hermaphrodites, indicating that AWA itself harbors sexual dimorphism. To ask how AWA function may be regulated by sex, we examined the response to da, which in hermaphrodites is mediated predominantly by AWA. We find that male da odortaxis is significantly lower than that of hermaphrodites and is not mediated by AWA. Consistent with this, expression of the da receptor ODR-10 is almost undetectable in male AWA neurons. Feminizing the male AWA neuron restores this expression, indicating that odr-10 is regulated by neural sex. Furthermore, the doublesex-like DM gene mab-3 is necessary for this sex difference, as mab-3 mutant males display significant ODR-10 expression in AWA. Together, these studies identify a mechanism by which cell-intrinsic sexual status regulates gene expression to control a sex difference in a shared sensory behavior.

Watabe, Eichi et al. (2019) International Worm Meeting "PES-4 regulates head-muscle-specific alternative splicing of the tropomyosin pre-mRNA."

KUROYANAGI, Hidehito, Watabe, Eichi

[International Worm Meeting, 2019]

In animals, many proteins involved in actin filament organization and functions have multiple isoforms owing to gene duplication and/or alternative pre-mRNA splicing. These isoforms are often expressed in cell-type-specific fashions. However, selection mechanisms and functional consequences of such protein isoforms in specific tissues largely remain to be elucidated. We have recently reported comprehensive analysis of splicing variants for the single tropomyosin gene lev-11 in C. elegans. We found that its novel alternative exon, 7a, is selected only in head muscles, whereas the other muscles and tissues select exon 7b (Mol Biol Cell, 2018; Cytoskeleton, 2018). Here we analyzed alternative splicing regulators of this head-muscle-specific splicing. We crossed dichromatic fluorescence lev-11 exon 7 splicing reporter worms with splicing factor mutants defective in muscle-specific alternative splicing and found that asd-2, a regulator for the let-2 gene and the unc-60 gene, are also involved at least in part in the head-muscle-specific splicing. We further searched recently published single-cell RNA-seq data for RNA-binding proteins enriched in the head muscles and identified pes-4, msi-1 and exc-7 as candidates. msi-1(os1) and exc-7(ok370) did not affect the lev-11 exon 7 splicing reporter. We then knocked out the pes-4 gene in the lev-11 exon 7 splicing reporter worms with CRISPR/Cas9 system and found that the pes-4 mutants completely lost the head-muscle-specific expression of exon 7a, although the homozygotes were arrested at the L1 stage. PES-4 protein has two KH-type RNA-binding domains, an alanine- and glutamine-rich (AQ-rich) region and a conserved C-terminal region. The C-terminal region matches the consensus of proline-tyrosine (PY)-type nuclear localization signal (PY-NLS) and were actually involved in the nuclear localization and function of PES-4. The AQ-rich region is not homologous to any other proteins in the primary sequence, but were essential for the splicing regulation. We are currently elucidating a model for exon 7a selection exclusively in the head muscles.