Antoine Donati

The evolution of complex brains required the incorporation of newly evolved neurons into existing circuits, yet the genetic and developmental mechanisms enabling their integration remain poorly understood. Butterflies expanded their color vision by adding a second R7 photoreceptor per ommatidium, a rare departure from the conserved insect eye ground plan. Because each R7 makes an independent stochastic fate choice, this duplication increased the number of ommatidial types from two to three. We identified the genetic basis of this expansion and demonstrated how the brain accommodates the new input. R7 duplication was achieved by converting R3/4 cells to an R1/6 fate, triggering the R7 recruitment program on both sides of the ommatidium. By manipulating transcription factor expression in Drosophila , we engineered a “butterfly-fly” that recapitulates this ancient R7 duplication and three-type stochastic mosaic. In the fly brain, two R7 subtypes connect to specific types of Dm8 neurons, which are born in excess and undergo apoptosis if they fail to find synaptic partners. In our butterfly-fly model, these surplus Dm8s are immediately rescued and form subtype-appropriate connections. These findings suggest that population-level variation maintains a reservoir of potentially interacting neurons, providing a developmental substrate that allows the brain to immediately accommodate newly evolved neurons.

The addition of new neuron types is thought to underlie the evolution of complex nervous systems, yet the mechanisms by which they arise remain unclear. Here we investigate the evolution of a novel target detection photoreceptor in the housefly Musca domestica. In males, a dorso-frontal eye region known as the Love Spot enables rapid detection and tracking of females during mating flights. In this region, R7 photoreceptors - normally dedicated to color vision - are repurposed for target detection through altered Rhodopsin expression, physiology, and circuit connectivity. We show that these Love Spot R7 cells (LsR7) are initially specified as canonical R7 photoreceptors but later adopt a chimeric identity combining transcription factors normally associated with either color or motion vision. The sex-determination transcription factor Doublesex (Dsx) is strongly upregulated after R7 specification and, together with the transcription factor Spineless, drives the LsR7 gene regulatory program. Because Dsx upregulation occurs after initial axon targeting to the medulla, LsR7 axons subsequently shorten to connect with lamina neurons and OFF-pathway motion vision circuits. These findings show how new combinations of cell-identity regulators can generate novel neural cell types while revealing developmental constraints imposed by evolutionary history.

The evolution of complex brains required existing neurons and neural circuits to accommodate new inputs. The genetic and developmental mechanisms that enable such integration are largely unknown. Butterflies evolved more complex retinal mosaics through the addition of a second R7 color photoreceptor per ommatidium (unit eye). In Drosophila , the unique R7 makes a stochastic choice to express one of two opsin genes. In butterflies, the two R7s make independent stochastic cell fate choices in each ommatidium, producing three ommatidial types instead of two. Here, we investigate the developmental basis of this change and how the butterfly brain accommodates expanded sensory receptor input. We first identified the changes in gene expression that cause a second R7 cell to be specified. We then modified Drosophila retinas to have butterfly-like transcription factor expression, causing recruitment of an additional R7. The two R7s make independent stochastic choices, like butterflies, leading to three stochastically distributed ommatidial types. In Drosophila , the two R7 subtypes connect to their target neurons, either yDm8 or pDm8. Dm8 neurons of both types are born in excess and Dm8s that do not find connections with their cognate y or pR7s undergo apoptosis. In the presence of extra R7s in butterfly-like fly retinas, additional Dm8s are retained, leading to two Dm8s per medulla column that make appropriate connections with the matching R7 subtypes, facilitating the expansion of color vision. We propose that the presence of cells that would otherwise die provide developmental flexibility that can allow brains to accommodate newly evolved inputs.

Body axis specification is a crucial event in animal embryogenesis and was an essential evolutionary innovation for founding the animal kingdom. This process involves two distinct components that coordinate to establish the spatial organisation of the embryo: initiation of cascades of regionalised gene expression and orientation of morphogenetic processes such as body elongation. Intense interest in the first component has revealed Wnt/β-catenin signalling as ancestrally responsible for initiating regional gene expression, but the evolutionary origin of oriented morphogenesis has received little attention. Here, by addressing the cell and morphological basis of body axis development in embryos of the cnidarian Clytia hemisphaerica, we have uncovered a simple and likely ancestral coordination mechanism between Wnt/β-catenin signalling and directed morphogenesis. We show that the ligand Wnt3, known to initiate oral gene expression via localised Wnt/β-catenin pathway activation, also has a key β-catenin-independent role in globally orienting planar cell polarity (PCP) to direct morphogenesis along the oral-aboral axis. This PCP orientation occurs in two distinct steps: local orientation by Wnt3 and global propagation by conserved core PCP protein interactions along the body axis. From these findings, we propose novel scenarios for PCP-driven symmetry-breaking underlying the emergence of the animal body plan.

Summary Body axis specification is a crucial event in animal embryogenesis and was an essential evolutionary innovation for founding the animal kingdom. It involves two distinct components that coordinate to establish the spatial organisation of the embryo: initiation of cascades of regionalised gene expression and orientation of morphogenetic processes such as body elongation. Intense interest in the first component has revealed Wnt/β-catenin signalling as ancestrally responsible for initiating regional gene expression, but the evolutionary origin of oriented morphogenesis has received little attention. Here, by addressing the cell and morphological basis of body axis development in embryos of the cnidarian Clytia hemisphaerica, we have uncovered a simple and likely ancestral coordination mechanism between Wnt/β-catenin signalling and directed morphogenesis. We show that the ligand Wnt3, known to initiate oral gene expression via localised Wnt/β-catenin pathway activation, also has a key β-catenin-independent role in globally orienting planar cell polarity (PCP) to direct morphogenesis along the oral-aboral axis. This PCP orientation occurs in two distinct steps: local orientation by Wnt3 and global propagation by conserved core PCP protein interactions along the body axis. From these findings we propose novel scenarios for PCP-driven symmetry-breaking underlying the emergence of the animal body plan.

A spectacular diversity of forms and features allow species to thrive in different environments, yet some structures remain relatively unchanged. Insect compound eyes are easily recognizable despite dramatic differences in visual abilities across species. It is unknown whether distant insect species use similar or different mechanisms to pattern their eyes or what types of genetic changes produce diversity of form and function. We find that flies, mosquitos, butterflies, moths, beetles, wasps, honeybees, and crickets use homologous developmental programs to pattern their retinas. Transcription factor expression can be used to establish homology of different photoreceptor (PR) types across the insects: Prospero (Pros) for R7, Spalt (Sal) for R7+R8, and Defective proventriculus (Dve) for R1-6. Using gene knockout (CRISPR/Cas9) in houseflies, butterflies, and crickets and gene knockdown (RNAi) in beetles, we found that like Drosophila , EGFR and Sevenless (Sev) signaling pathways are required to recruit motion and color vision PRs, though Drosophila have a decreased reliance on Sev signaling relative to other insects. Despite morphological and physiological variation across species, retina development passes through a highly conserved phylotypic stage when the unit eyes (ommatidia) are first patterned. This patterning process likely represents an “insect eye ground plan” that is established by an ancient developmental program. We identify three types of developmental patterning modifications (ground plan modification, nonstochastic patterns, and specialized regions) that allow for the diversification of insect eyes. We suggest that developmental divergence after the ground plan is established is responsible for the exceptional diversity observed across insect visual systems.

Summary: Body axis specification is a crucial event in animal embryogenesis and was an essential evolutionary innovation for founding the animal kingdom. It involves two distinct components that coordinate to establish the spatial organisation of the embryo: initiation of cascades of regionalised gene expression and orientation of morphogenetic processes such as body elongation. Intense interest in the first component has revealed Wnt/β-catenin signalling as ancestrally responsible for initiating regional gene expression, but the evolutionary origin of oriented morphogenesis has received little attention. Here, by addressing the cell and morphological basis of body axis development in embryos of the cnidarian Clytia hemisphaerica, we have uncovered a simple and likely ancestral coordination mechanism between Wnt/β-catenin signalling and directed morphogenesis. We show that the ligand Wnt3, known to initiate oral gene expression via localised Wnt/β-catenin pathway activation, also has a key β-catenin-independent role in globally orienting planar cell polarity (PCP) to direct morphogenesis along the oral-aboral axis. This PCP orientation occurs in two distinct steps: local orientation by Wnt3 and global propagation by conserved core PCP protein interactions along the body axis. From these findings we propose novel scenarios for PCP-driven symmetry-breaking underlying the emergence of the animal body plan.

Epithelial cilia, whether motile or primary, often display an off-center planar localization within the apical cell surface. This form of planar cell polarity (PCP) involves the asymmetric positioning of the ciliary basal body (BB). Using the monociliated epithelium of the embryonic zebrafish floor-plate, we investigated the dynamics and mechanisms of BB polarization by live imaging. BBs were highly motile, making back-and-forth movements along the antero-posterior (AP) axis and contacting both the anterior and posterior membranes. Contacts exclusively occurred at junctional Par3 patches and were often preceded by membrane digitations extending towards the BB, suggesting focused cortical pulling forces. Accordingly, BBs and Par3 patches were linked by dynamic microtubules. Later, BBs became less motile and eventually settled at posterior apical junctions enriched in Par3. BB posterior positioning followed Par3 posterior enrichment and was impaired upon Par3 depletion or disorganization of Par3 patches. In the PCP mutant vangl2, BBs were still motile but displayed poorly oriented membrane contacts that correlated with Par3 patch fragmentation and lateral spreading. Thus, we propose an unexpected function for posterior Par3 enrichment in controlling BB positioning downstream of the PCP pathway.