Conserved nuclear hormone receptors controlling a novel plastic trait target fast-evolving genes expressed in a single cell

Abstract

Environment shapes development through a phenomenon called developmental plasticity. Deciphering its genetic basis has potential to shed light on the origin of novel traits and adaptation to environmental change. However, molecular studies are scarce, and little is known about molecular mechanisms associated with plasticity. We investigated the gene regulatory network controlling predatory vs. non-predatory dimorphism in the nematode Pristionchus pacificus and found that it consists of genes of extremely different age classes. We isolated mutants in the conserved nuclear hormone receptor nhr-1 with previously unseen phenotypic effects. They disrupt mouth-form determination and result in animals combining features of both wild-type morphs. In contrast, mutants in another conserved nuclear hormone receptor nhr-40 display altered morph ratios, but no intermediate morphology. Despite divergent modes of control, NHR-1 and NHR-40 share transcriptional targets, which encode extracellular proteins that have no orthologs in Caenorhabditis elegans and result from lineage-specific expansions. An array of transcriptional reporters revealed co-expression of all tested targets in the same pharyngeal gland cell. Major morphological changes in this gland cell accompanied the evolution of teeth and predation, linking rapid gene turnover with morphological innovations. Thus, the origin of feeding plasticity involved novelty at the level of genes, cells and behavior.

Fig 1. Mouth-form plasticity in P. pacificus.

(A) Mouth structure of wild-type eurystomatous (Eu) morph, wild-type stenostomatous (St) morph, nhr-1 mutant, and nhr-40 mutant. Unlabeled images in two focal planes are shown in S1A Fig. (B) Scanning electron microscopy image of the mouth opening of the Eu morph. (C) The Eu morph devouring its prey. (D) Putative gene regulatory network controlling mouth-form plasticity in P. pacificus. (E) Design of the suppressor screen. DT = dorsal tooth, RVSLT = right ventrosublateral tooth, RVSLR = right ventrosublateral ridge, EMS = ethyl methanesulfonate.

Fig 2. Reverse genetics, transcriptomics and expression patterns of nhr-40 and nhr-1.

(A) Protein structure of NHR-40 in wild-type and mutant animals. (B) Expression levels of nhr-40 and nhr-1 in wild type and mutants as revealed by transcriptomic profiling. (C) Antibody staining against the HA epitope in an nhr-1 rescue line. (D) Expression patterns of nhr-40 and nhr-1 transcriptional reporters in a double reporter line. TurboRFP (magenta) and Venus (green) channels are presented as maximum intensity projections. Co-expression results in white color. D = dorsal, V = ventral, A = anterior, P = posterior, N.S. = not significant, FPKM = Fragments Per Kilobase of transcript per Million mapped reads.

Fig 3. Target genes of NHR-40 and NHR-1.

(A) Experimental setup of transcriptomics experiment and selection criteria to identify target genes. (B) Trends among target genes compared to genome-wide pattern. (C) Transmission electron microscopy reconstruction of the dorsal pharyngeal gland cell (g1D) [53] and expression patterns of transcriptional reporters for nine selected targets of NHR-40 and NHR-1. TurboRFP channel is presented as standard deviation projections. lof = loss-of-function, gof = gain-of-function, *** = p<0.001, D = dorsal, V = ventral, A = anterior, P = posterior.

Fig 4. Evolution of pharynx morphology in the order Rhabditida.

Fig 5. Evolution of nhr-40, nhr-1, and their target genes.

Arrowheads point at the genes of interest. Protein-based trees of NHR genes (A), Astacin domains (B), chitinase domains (C), and CAP domains (D) in P. pacificus and C. elegans. (E) Nucleotide-based tree of the CAP domains from a poorly-resolved protein-based subtree of all predicted CAP domains in P. pacificus and P. fissidentatus.