Development of functional ectopic compound eyes in scarabaeid beetles by knockdown of orthodenticle

Abstract

Complex traits like limbs, brains, or eyes form through coordinated integration of diverse cell fates across developmental space and time, yet understanding how complexity and integration emerge from uniform, undifferentiated precursor tissues remains limited. Here, we use ectopic eye formation as a paradigm to investigate the emergence and integration of novel complex structures following massive ontogenetic perturbation. We show that down-regulation via RNAi of a single head patterning gene-orthodenticle-induces ectopic structures externally resembling compound eyes at the middorsal adult head of both basal and derived scarabaeid beetle species (Onthophagini and Oniticellini). Scanning electron microscopy documents ommatidial organization of these induced structures, while immunohistochemistry reveals the presence of rudimentary ommatidial lenses, crystalline cones, and associated neural-like tissue within them. Further, RNAsequencing experiments show that after orthodenticle downregulation, the transcriptional signature of the middorsal headthe location of ectopic eye induction-converges onto that of regular compound eyes, including up-regulation of several retina-specific genes. Finally, a light-aversion behavioral assay to assess functionality reveals that ectopic compound eyes can rescue the ability to respond to visual stimuli when wild-type eyes are surgically removed. Combined, our results show that knockdown of a single gene is sufficient for the middorsal head to acquire the competence to ectopically generate a functional compound eye-like structure. These findings highlight the buffering capacity of developmental systems, allowing massive genetic perturbations to be channeled toward orderly and functional developmental outcomes, and render ectopic eye formation a widely accessible paradigm to study the evolution of complex systems.developmental buffering | complex trait evolution | ex situ development | organoid | RNA-seq D evelopmental systems channel tissues with broad potential into tightly regulated, specific fates, thereby enabling the formation of highly complex traits like limbs, brains, photic organs, or eyes. How complex traits develop from undifferentiated precursor tissues and how this ability itself originated and diversified are questions that have motivated biologists since the emergence of evolutionary thinking. The evolution and development of arthropod compound eyes in particular has attracted significant attention due to the high level of structural and regulatory complexity required to produce a functional organ: Functional compound eyes exhibit ommatidial organization, possess neural projections connecting to the central nervous system, express eye development and photoreceptor genes, and enable an integrated behavioral response to light stimuli. Insect eyes develop from pluripotent anterior head regions also capable of generating single-lens ocelli and nonvisual epidermis. Patterning of this region, best understood in Drosophila flies and Tribolium beetles, depends on a complex a...

Chromoanagenesis: a piece of the macroevolution scenario

“…More anecdotal, the recent discovery of a python with a third functioning eye on its forehead could also be related to the macroevolution process (https://www.livescience.com/65382-three-eyedsnake.html). Similar ectopic induction of functional eyes has been genetically induced in scarab beetles [60]. The rapid emergence and integration of novel complex organs such as a eye or an extra finger provide remarkable examples of the ability of developmental systems to channel massive perturbations toward orderly and functional outcomes, highlighting the extraordinary plasticity of genome and the buffering capacity of developmental systems.…”

Integrating evolutionarily novel horns within the deeply conserved insect head

“…In a follow-up study, Zattara et al were able to show that one of the patterning genes critical for establishing said boundary during embryogenesis-otdhas been repurposed during late larval development to pattern the medial dorsal head. otd RNAi phenotypes reported by Zattara et al included a reduction or loss of posterior head horns, the expression of ectopic horns in more anterior locations, and-curiously-the induction of ectopic medial compound eyes [11,37]. Collectively, the results of these two studies motivated the suspicion that the positioning and integration of horns within the posterior head may be enabled by the repurposing of genes and developmental processes already involved in patterning the corresponding regions during earlier developmental stages, and in addition may to some degree involve network components ancestrally associated with the induction and positioning of eyes including ocelli.…”

Wnt controls the medial–lateral subdivision of theDrosophilahead

“…Recent work by Zattara et al . [ 25 ] found that RNAi-mediated otd attenuation in Onthophagus beetles resulted in the development of CEs on the dorsal head. These authors acknowledge that most beetle families have lost ocelli and speculate that otd attenuation might have triggered the atavistic ocellar program expressed as CE [ 25 ].…”

“…Therefore, medial head identity would be regulated by wg through two mechanisms: establishing an otdþ/ey2 domain where ocelli develop, and by preventing dpp expression and/or signalling (figure 2g; see [10,24]). Recent work by Zattara et al [25] found that RNAi-mediated otd attenuation in Onthophagus beetles resulted in the development of CEs on the dorsal head. These authors acknowledge that most beetle families have lost ocelli and speculate that otd attenuation might have triggered the atavistic ocellar program expressed as CE [25].…”