Littleford, Hana et al. (2019) International Worm Meeting "bHLH genes control cell fate in the sexually dimorphic somatic gonad."

Greenwald, Iva, Finkelstein, Sarah, Littleford, Hana

[International Worm Meeting, 2019]

Many aspects of the sexually dimorphic form and function of the developing C. elegans somatic gonad are governed by differentiated regulatory cells. In hermaphrodites, the distal tip cells (hDTCs) lead outgrowth of the two gonad arms and act as a germline niche while the anchor cell (AC) organizes vulval and uterine development. In males, the mDTCs act as the niche and the linker cell (LC) leads outgrowth of the single gonad arm. We have previously shown that each regulatory cell type expresses a unique complement of basic helix-loop-helix (bHLH) transcription factors, and proposed that a "bHLH code" specifies the fates and functions of the regulatory cells (Sallee et al. 2017). The bHLH code hypothesis posits that the inputs of sex, developmental stage, lineage, and timing leads to the expression of the appropriate bHLH complement in each prospective regulatory cell. In support of this hypothesis, we were able to reprogram the AC to assume an mDTC fate by ectopically expressing the mDTC bHLH code. We have now performed further bHLH complement manipulations and observed AC?LC and AC?hDTC cell fate transformations, and partial LC?AC transformation, consistent with the bHLH code hypothesis. To investigate sex as an input to gonadal regulatory cell specification, we are observing how regulatory cell fates are altered in sexual differentiation mutants. fkh-6(0) XO mutants have an AC (Chang et al. 2004), indicating a feminized proximal gonad, but we find that the distal regulatory cells express an mDTC marker, suggesting that other inputs specify their male identity. Finally, the different adult morphology of somatic gonads of other nematode species may reflect evolution of regulatory cell roles (Felix and Sternberg 1996). To investigate if bHLH genes control these differences, we have identified putative orthologs of the bHLH code genes in most other nematodes. Strikingly, hlh-12, which regulates migration of hDTCs and the LC in C. elegans (Tamai and Nishiwaki 2007), is not conserved in other nematodes. We plan to identify additional candidate upstream factors by searching for regulatory elements in bHLH genes that regulate their cell type-specific expression in C. elegans, and to explore bHLH expression and function in other species.

Yang, Ji-Sup et al. (2015) International Worm Meeting "Identification of DMD-3 targets in the C. elegans male tail tip."

Williams, Emily, Herrera, R Antonio, Ercan, Sevinc, Kiontke, Karin, Yang, Ji-Sup, Fitch, David, Mason, Adam, Littleford, Hana

[International Worm Meeting, 2015]

Cellular morphogenesis is a fundamental developmental mechanism that governs how cells migrate, change identity, change shape, and fuse. The C. elegans male tail tip undergoes a morphogenetic event in just four cells during late L4, making this a powerful model for elucidating linkage between regulators of timing, position, and epidermal fate and the cell-biological mechanisms required for morphogenesis. Male tail tip morphogenesis (TTM) is governed by DMD-3, a conserved DM-domain transcription factor that is homologous to DMRT1, which regulates male fate in humans. Expression of dmd-3 is necessary and sufficient for TTM to occur (Mason et al. 2008). Previous work done in the lab has shown that DMD-3 is at the center of a genetic network with a bow-tie structure, regulated by pathways including Wnt signaling and heterochronic regulation, and in turn regulating genes involved in cellular processes such as vesicle trafficking and cytoskeletal rearrangement (Nelson et al 2011). However, while some of the regulatory pathways involved in specifying DMD-3 expression are known, the structure of the network downstream of DMD-3 is uncharacterized. We are using two methods to investigate downstream targets of DMD-3: To identify tail tip-specific targets, we are performing transcriptome profiling of isolated tail tip tissue from worms that undergo tail tip morphogenesis (wild-type males and hermaphrodites ectopically expressing dmd-3), and worms that do not (wild-type hermaphrodites and dmd-3 mutant males). To identify direct DMD-3 transcriptional targets, we are employing chromatin immunoprecipitation and next-generation sequencing (ChIP-Seq), utilizing a CRISPR-generated dmd-3 allele tagged with GFP and 3xFLAG. Preliminary microarray transcriptome analysis on tail tip-specific RNA identified 134 significantly differentially expressed genes in the dmd-3 mutant relative to wild-type. Combining the results of both approaches will yield direct targets of DMD-3 that act in the male tail tip.References: Mason et al. 2008. Development 135:2373-2382. Nelson et al. 2011. PLoS Genetics 7(3):e1002010.

Rhonda Hyde et al. (2003) International Worm Meeting "Identifying candidate genes involved in serotonin modulation of a C. elegans behavioral circuit"

Rhonda Hyde, Pierre-Olivier Barome, Anne C Hart, Laurent Segalat, Michael Y Chao

[International Worm Meeting, 2003]

In the nervous system, G-protein coupled receptors (GPCRs) mediate slow synaptic transmission through the activation of signaling cascades. GPCRs have been shown to alter synaptic transmission both presynaptically by affecting neurotransmitter release and postsynaptically by modulating receptor function and localization. Work in our lab has shown that avoidance of the chemical repellant 1-octanol is modulated by serotonin. Laser ablation experiments reveal that well-fed animals sense the presence of octanol through the ASH sensory neurons while octanol detection in starved animals involves ASH and two other pairs of sensory neurons, ADL and AWB (1). This food-dependent effect can be mimicked with exogenous serotonin, consistent with increased levels of serotonin in animals on food. These studies show that feeding status and endogenous serotonin levels are responsible for selective signaling by sensory neurons in octanol avoidance. We are interested in identifying the serotonin GPCRs that modulate octanol avoidance to determine the sites of action of serotonin in the avoidance circuit. There are 2 characterized C. elegans serotonin GPCRs, 5-HT-Ce (2) and 5-HT2-Ce (3). From a search of the C. elegans genome, we identified 10 additional receptors that share sequence similarity to the 5-HT-Ce receptor. Transcriptional fusion constructs will be used to determine the expression patterns of each receptor. Receptors that are expressed in either the sensory neurons or command interneurons are strong candidates for modulating the octanol avoidance response. RNAi will be used to determine their functional significance in avoidance behavior. A previous screen conducted by Hana Fukuto in the lab identified mutant strains with defective octanol responses off food but normal responses on food. Exogenous serotonin restores normal response to octanol in all but one strain. This mutant strain also has defects in other serotoninergic behaviors. We are mapping and further characterizing the corresponding gene to determine its role in serotonin signaling. References: (1) Chao et al ECWM 2002, IWM 2003 (2) Olde and McCombie J Mol Neurosci 1997 (3) Hamdan et al J. Neurochem 1999