A sex-specific regulator expands the embryonic ocular field to generate a novel visual system

Technical Summary: A Sex-Specific Regulator Expands the Embryonic Ocular Field to Generate a Novel Visual System

Problem and Context
The origin of morphological novelties is a central question in evolutionary developmental biology. While new organs often arise through the redeployment of conserved Gene Regulatory Networks (GRNs) into novel developmental contexts, the specific genetic and genomic changes that trigger these co-option events remain poorly understood. This is particularly true for sexually dimorphic innovations where sex-determination pathways interact with organ-specification networks. The mayfly Cloeon dipterum (Baetidae) presents a unique case: males possess a male-specific, additional visual system known as Turbanate Eyes (TurbEyes), consisting of a pair of large dorsal compound eyes and dedicated optic lobes. This structure is absent in females, who retain the ancestral insect visual system (lateral compound eyes and ocelli). Unlike many insect head novelties which are simple outgrowths, TurbEyes represent a complex, functional sensory system. The genetic mechanisms governing the origin and development of this male-specific trait were previously unexplored.

Methodology
The authors employed a multi-omics approach combined with functional genetics in C. dipterum:

1. Single-Cell Transcriptomics (scRNA-seq): Using SPLiT-seq with ACME fixation to minimize dissociation artifacts, the authors profiled heads from three nymphal stages (NIII, NV, NVII). They generated specific male and female samples for stages NV and NVII, where TurbEye primordia are visible, while NIII samples were mixed sexes.
2. Chromatin Accessibility Profiling (ATAC-seq): Performed on dissected heads from corresponding developmental stages to map accessible regulatory regions.
3. Computational Integration: The authors integrated scRNA-seq and ATAC-seq data using a customized SCENIC pipeline to reconstruct Gene Regulatory Networks (GRNs) and identify active regulons at single-cell resolution. They also utilized ANANSE to infer regulatory networks from embryonic bulk RNA-seq and ATAC-seq data.
4. Spatial Validation: Hybridization Chain Reaction (HCR) and standard in situ hybridization were used to visualize gene expression patterns in embryonic and nymphal tissues.
5. Functional Genetics:
   • Parental RNAi: Injection of dsRNA against candidate genes into gravid females to knock down gene function during embryogenesis.
   • DIPA-CRISPR: Direct injection of CRISPR/Cas9 components into fertilized females to generate mosaic loss-of-function individuals for phenotypic analysis in nymphs and adults.

Key Results

1. Deployment of the Ancestral RDGN: Single-cell analysis revealed that the developing TurbEye and its associated optic lobes express the full suite of the Retinal Determination Gene Network (RDGN), including eyeless/Pax6 (ey), sine oculis (so), optix, dachshund (dac), and eyes absent (eya). This indicates that the TurbEye arises not from a lineage-specific network, but through the spatial redeployment of the conserved ancestral retinal program.
2. Identification of dsxM: Differential expression analysis between male and female visual tissues identified a novel, male-specific paralog of the sex-determination transcription factor doublesex, named dsxM. Phylogenetic analysis confirmed dsxM as a third dsx paralog specific to C. dipterum. dsxM expression is restricted to the developing TurbEye retina and optic lobes in males and is absent in females and in the lateral eyes of males.
3. Regulatory Hierarchy: Network inference (SCENIC and ANANSE) positioned dsxM as a top-level "initiator" regulator. The data predicted that dsxM directly activates eyeless (ey). Chromatin accessibility analysis confirmed the presence of predicted Dsx-binding motifs within accessible regions of the ey locus.
4. Functional Validation:
   • Embryonic Expansion: In control male embryos, the ey expression domain in the dorsal procephalon is significantly expanded compared to females. Knockdown of dsxM via parental RNAi resulted in a significant reduction of this male-specific ey expansion, causing the domain to shrink to dimensions similar to those in females.
   • TurbEye Morphogenesis: Mosaic CRISPR individuals with localized dsxM disruption exhibited clear defects in TurbEye morphology, including abnormal ommatidial organization. This suggests dsxM is required not only for the initial establishment of the expanded ocular field but also for subsequent TurbEye morphogenesis.

Key Contributions

• Mechanism of Novelty: The study demonstrates that a morphological novelty (the TurbEye) can be generated by the expansion of a pre-existing developmental field (the embryonic ocular territory) rather than the invention of new cell types or networks.
• Sex-Specific Co-option: It identifies a specific molecular mechanism where a sex-determination factor (dsxM) co-opts a master regulator of organ identity (ey) to create a new developmental territory.
• Evolutionary Insight: The findings reveal that the activation of a single upstream regulator (ey) in a novel cephalic location, driven by a sex-specific paralog, is sufficient to trigger the deployment of the entire conserved retinal developmental program.

Significance and Claims
The authors claim that their findings provide a mechanistic example of how conserved developmental programs are redeployed in evolution. Specifically, they show that a sex-specific regulator (dsxM) can expand a pre-existing developmental field, enabling the spatial redeployment of an ancestral GRN (the RDGN) to generate a novel, male-specific visual system. This work bridges the gap between sex-determination pathways and organ-specification networks, suggesting that sexual dimorphism can drive morphological innovation by modifying the spatial extent of conserved developmental programs. The TurbEye serves as a model for understanding how new developmental territories are created, allowing for subsequent structural and functional diversification without the need for taxon-restricted genes.