# Expanding the fly eye gene regulatory network: From Drosophila to the hoverfly Episyrphus balteatus

## TL;DR

The study compares eye development gene networks in fruit flies and hoverflies, revealing conserved genes and regulatory connections that evolved differently over millions of years.

## Contribution

The paper introduces the hoverfly Episyrphus balteatus as a model to expand and compare the eye gene regulatory network with Drosophila, identifying conserved and divergent regulatory interactions.

## Key findings

- A core set of 22 transcription factors is conserved between Drosophila and Episyrphus eye GRNs.
- Lozenge (lz) was experimentally confirmed as a negative regulator of dachshund (dac) in Drosophila.
- Regulatory link conservation varies across genes, suggesting extensive network rewiring over evolutionary time.

## Abstract

Comparing organ-specific gene regulatory networks (GRNs) across large evolutionary distances remains a major challenge, particularly when the species under study differ in data resolution. This applies to the GRN controlling compound eye development in insects, which is well characterized in Drosophila melanogaster but less well understood in other lineages. Here, we introduce the marmalade fly Episyrphus balteatus (Syrphidae), which diverged from Drosophila approximately 90 million years ago, as a comparative model to study eye development in Diptera. Using RNA-seq and ATAC-seq datasets, we reconstruct the first eye GRN in Episyrphus. Many genes involved in early Drosophila eye specification and differentiation are also active in Episyrphus. Among these, both species share a set of 22 transcription factors (TFs). The GRN built from these TFs and their DNA-binding motifs displays a high degree of internal connectivity. Link conservation analysis, followed by experimental testing in Drosophila, further identifies the AML1/Runx transcription factor lozenge (lz) as a negative regulator of the retinal determination gene dachshund (dac). Within the eye GRN, the degree of regulatory link conservation varies among genes, and both the number and position of regulatory regions often differ across orthologous loci. These results expand the eye GRN of Diptera and suggest extensive network rewiring over the evolutionary span separating Episyrphus and Drosophila.

The gene regulatory network (GRN) controlling the specification and early patterning of the Drosophila eye is often used as a reference for studying insect compound eyes. However, Drosophila belongs to a derived lineage of flies, and it remains unclear which aspects of its eye GRN are broadly conserved and which are lineage-specific. To identify genes and regulatory interactions conserved beyond Drosophila melanogaster and its close relatives, we generated the eye GRN of Episyrphus balteatus, a hoverfly that diverged from Drosophila approximately 90 million years ago, and compared it with the Drosophila eye GRN using RNA-seq and ATAC-seq data. This comparison reveals a core set of conserved genes, including 22 shared transcription factors. The resulting networks show a high degree of connectivity, although the conservation of individual regulatory links varies among genes. We further tested the predictive power of conserved links by examining the relationship between the AML-family transcription factor Lozenge (Lz) and the retinal gene dachshund (dac), and found that dac is indeed regulated by lz. Overall, our work expands the Dipteran eye GRN and suggests that network connectivity is evolutionarily fluid, allowing extensive rewiring over long evolutionary timescales.