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Science AdvancesDevelopmental BiologyPeer-reviewed journal article

A panoramic view of the expression and function of the Doublesex/DMRT gene family in C. elegans

Chen WangiD, Yehuda SalzbergiD, Meital Oren‐SuissaiD, Oliver HobertiD

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Abstract

Throughout the animal kingdom, sex determination and sexual differentiation are orchestrated by a strikingly diverse set of regulatory factors. The only type of molecules consistently deployed during sexual differentiation are members of the Doublesex/Mab-3-related transcription factor (DMRT) family. Although each animal genome codes for a multitude of DMRT family members, in no species has the full array of DMRT genes been comprehensively analyzed across the entire animal, in all sexes and throughout development. Hence, the extent of deployment of DMRT genes in sexual differentiation remains unknown. We describe here the first genome- and nervous system-wide expression and functional analysis of all members of the DMRT gene family. Leveraging genome-engineered reporter alleles of all ten DMRT genes of the nematode Caenorhabditis elegans, we find that six DMRTs display sexually dimorphic expression in somatic and/or reproductive tissues, including in cell and tissue types not previously known to be sexually dimorphic. In the nervous system, DMRT protein expression covers many, though not all, known sexually dimorphic neuron types. Analyses of DMRT null mutant alleles reveal a suite of neuronal differentiation defects, ranging from altered neurotransmitter identities and switched neuropeptide signatures to impaired glia-to-neuron transdifferentiation. Several DMRT proteins do not exhibit sexually dimorphic expression, indicating roles beyond sexual differentiation. Similar comprehensive analyses of DMRT genes in other organisms may help to better understand the extent and regulation of sex-specific cellular differentiation programs.

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (5 Mar 2026) · CC BY 4.0

Reporter alleles of all ten C. elegans DMRT genes show six are sexually dimorphic across tissues; mab-3, dmd-3 and mab-23 nulls alter male neuronal identities.

DMRT genes are the one gene family consistently used in sexual differentiation across animals, yet no species had a whole-animal, both-sex, all-stage analysis of the full family. This work supplies that protein-level atlas in C. elegans and tests function in the male nervous system.

Key results

  1. ShownSix of the ten DMRT proteins are sexually dimorphic in adults, with dimorphism in every major cell type, including cell types not previously known to be dimorphic.

    Six of ten dimorphic. MAB-3 is male-specific in pharyngeal muscle, pharyngeal-intestinal valve cells and the excretory canal cell; MAB-23 and DMD-3 are dimorphic in body wall muscle. DMD-5, DMD-6 and DMD-8 show no dimorphism. · Six C. elegans DMRT proteins reveal sexual…; Sexually dimorphic expression of DMRT proteins…; Non-sexually dimorphic expression of DMRT…

  2. ShownDMRT proteins mark sexual dimorphism in a large fraction of sex-shared neurons, mostly male-enriched, with MAB-3 contributing much of it.

    Of 294 sex-shared neurons, 163 express DMRTs cell-specifically (excluding DMD-6 and DMD-7); 143 (~49%) express at least one DMRT in one sex only. This is 44 of 116 sex-shared neuron classes. · DMRT proteins reveal extensive sexual…

  3. ShownMost male-specific neurons express a DMRT protein; hermaphrodite-specific neurons express none. Co-expression subdivides anatomical neuron classes (e.g. CA, CP) into molecular subclasses.

    69 of 93 (74%) male-specific neurons express DMRTs, covering 21 of 25 classes; 0 of 8 hermaphrodite-specific neurons do. 22 of the 69 express more than one DMRT, most often DMD-3 plus MAB-23. · DMRT proteins are expressed in most, but not…

  4. ShownNull mutants of mab-3, dmd-3 and mab-23 alter cholinergic, glutamatergic and monoaminergic identities in many male-specific neurons, with no GABAergic changes and neuron generation largely intact, except mab-3 effects on V lineage ray neurons.

    Among 69 male-specific neurons expressing these DMRTs, 30 show altered identities, spanning 10 of 21 DMRT-expressing classes. 15-30 animals scored per strain; Fisher's exact, Mann-Whitney or Kruskal-Wallis tests. · DMRT genes are broadly required for conferring…; DMRT genes broadly regulate neuronal…; Neuronal identity mutant analysis; Figure legends

  5. Shownmab-3 acts as a bidirectional regulator of neuropeptide identity in AWA and is needed for glia-to-neuron transdifferentiation of PHso1 into PHD in males.

    In mab-3 null males, AWA gains flp-20 expression while nlp-15 expression is reduced. Males fail to form the anterior PHD axon, retain socket-like PHso1 structures, and show strongly reduced oig-8p::gfp. · A DMRT gene is required for a sex-specific…; A DMRT gene is required for sex-specific…; Figure legends

Methods, data and reproducibility

Endogenous fluorescent reporter alleles of the ten DMRT genes were imaged in both sexes across development, with NeuroPAL for neuron identification. Null alleles of mab-3, dmd-3 and mab-23 were crossed into neurotransmitter and neuropeptide reporters and scored.

  • CRISPR/Cas9 endogenous reporter alleles of all ten DMRT genes imaged in both sexes across larval stages and adults · Caenorhabditis elegans
  • Null mutants of mab-3, dmd-3 and mab-23 crossed into neurotransmitter and neuropeptide reporter strains in the NeuroPAL background · Caenorhabditis elegans · n = 15-30 animals scored per strain
  • dmd-10 full-locus null mutant tested for male mating behavior, with PHB rescue and synapse-labeling assays · Caenorhabditis elegans
  • Comparative DMRT gene complements in other nematodes (C. remanei, P. pacificus, O. volvulus, P. sambesii) · multiple nematode species · n = 19 DMRT genes in P. pacificus
Methods & tools
CRISPR/Cas9 genome engineering, NeuroPAL landmark strain, Zeiss LSM880 and LSM980 confocal microscopy, ZEN software, GraphPad Prism version 10, Fisher's exact test, Kruskal-Wallis test with Dunn's multiple comparison, Mann-Whitney test, GRASP and iBlinc synapse labeling, WormBase genome annotation
Reagents & environment
GraphPad Prism version 10 for statistics; ZEN software for image processing and intensity quantification.
Reproducibility gaps
No code repository stated. New strains deposited at the CGC; strain lists and primer/sgRNA sequences are in supplementary Data S1 and S2.
Limitations (5)
  • Reporter alleles may yield expression below detectability, so absence of signal (e.g. DMD-7 in some neurons, AVG for DMD-5 and DMD-10) does not rule out low-level expression. · stated by the authors
  • Autofluorescence in the spermatheca and seminal vesicle means DMD-9 signal there cannot be distinguished from background. · stated by the authors
  • Reporter and neuron-identification differences likely explain why R3A and R4A identity changes reported earlier were not reproduced here. · stated by the authors
  • Functional tests cover only mab-3, dmd-3, mab-23 and dmd-10; other DMRT genes were not assessed, and many scores are qualitative (on/off/dim) in 15-30 animals. · noted in this summary
  • Links from MAB-3 or MAB-23 expression to pharyngeal pumping, body-wave speed or osmotic tolerance are speculative; no functional data are presented. · noted in this summary

Read this if you study sexual dimorphism, neuronal identity specification or Doublesex/DMRT biology and want a cell-resolution, protein-level expression atlas with mutant phenotypes in C. elegans.

Citations
1
FWCI
3.96
Field percentile
top 9%

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