Andrew Tomlinson

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 fly eye is composed of hundreds of ommatidia, each containing a stereotyped pattern of photoreceptors and assorted support cells. Over the past 50 years, molecular genetic studies have illuminated how the fates of many of these cells are specified. As a result, the field has become a classic paradigm for cell-fate specification during animal development. The initial focus of the studies was the sevenless (sev) mutant, in which the cell normally destined to become the R7 photoreceptor instead develops into a lens-secreting support cell. This phenotype served as the entry point into the patterning mechanisms underlying ommatidial fate specification. sev was found to encode a receptor tyrosine kinase (RTK) expressed on the plasma membrane of the R7 precursor. Its ligand—Bride of Sevenless (Boss)—was then identified as a plasma membrane protein expressed on a neighboring cell, the presumptive R8 photoreceptor. Here, I present a personal perspective on the studies of Sev, from its discovery to the present day, beginning with the characterization of its loss-of-function phenotype and culminating in the highly detailed description of its biological role and the molecular mechanism by which it transduces the R8-to-R7 Boss signal. These studies have revealed surprisingly complex and novel properties of this seemingly simple signaling mechanism, including (i) the unexpected circuitry by which Sev signaling is integrated with that of the Drosophila EGF receptor and Notch to specify 3 distinct cell types (R7 and R1/6 photoreceptors and the lens-secreting cone cells) and (ii) the likely discovery of a new molecular mechanism of RTK activation in which transendocytosis of Boss from the presumptive R8 into the R7 precursor directs Sev to a select endocytic compartment, where it is activated by a conformation state induced by low pH. Further, I address evolutionary studies of Sev and Boss, which indicate that their roles as ligand and receptor are restricted to flies, and pose the intriguing question of whether R7 photoreceptors outside Diptera are specified by similar signaling logic but different molecular machinery.

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.

When a cell receives multiple developmental signals simultaneously, the intracellular transduction pathways triggered by those signals are coincidentally active. How then, do the cells decode the information contained within those multiple active pathways to derive a precise developmental directive? The specification of the Drosophila R7 photoreceptor is classic model system for investigating such questions. The R7 fate is specified by the combined actions of the of the Notch (N) and receptor tyrosine kinase (RTK) signaling pathways. The two pathways cross-communicate in an integrative mechanism but also supply information independently of each other. Collectively, this information is summed to provide an unambiguous directive for the R7 fate. Our goal is to understand these mechanisms. Here, we examine how N activity represses transcription of the phyllopod gene in the process of information integration with the RTK pathway, and how it represses expression of the seven-up gene in an independent mechanism needed for R7 fate. We describe how N activity achieves these transcriptional repressions, and identify Enhancer of Split transcription factors as the mediators of those actions. Summary Statement: This work examines the role played by Enhancer of Split transcription factors in mediating N signals in Drosophila R7 photoreceptor specification.

The Drosophila R7 photoreceptor is a classic model for understanding how integration of signaling information can provide precise fate directives. It entails extensive interactions between the RTK and Notch signaling pathways, with Notch providing three distinct functions: it both opposes and promotes the general photoreceptor fate, and it determines the specific photoreceptor type. The RTK pathway promotes transcription of phyl - a gene expression critical for photoreceptor specification. We show that Notch activity induces transcription of yan which encodes a transcriptional repressor of phyl . This defines the antagonism between the two pathways, with RTK promoting and Notch opposing phyl transcription. We previously showed that Notch provides Sevenless to the cell to allow RTK pathway hyperactivation to overcome the Notch repression, and we now identify the regulation of Yan activity as the integration site of the RTK and Notch functions. Once the cell is specified as a photoreceptor, the third Notch function then prevents seven-up ( svp ) transcription. The Svp transcription factor directs the R1/6 photoreceptor fate, and the prevention of its expression ensures the default R7 specification. Summary Statement: This paper examines how the different signals received by the Drosophila R7 photoreceptor precursor are decoded and used to direct the cell’s appropriate differentiation pathway.

In Planar cell polarity (PCP), cells coordinately polarize their cytoskeletons within the plane of the epithelium in which they lie. In most insect epithelia this is indicated by the coordinated projections of the hairs secreted by the ectodermal cells. PCP of this form has been effectively studied in Drosophila, but it has proven difficult to achieve an integrated description of the roles played by the various proteins. In the insect eye, PCP is not evident as the polarization of individual cells, but as the asymmetric arrangements of the cells of the ommatidia. This different form of PCP allows different studies to be performed, and using this system we have detected the action of two antagonistic signaling pathways. Even though antagonistic, the two pathways synergize and cooperate to ensure that the correct arrangement of the cells is achieved. The cooperative use of antagonistic signaling pathways occurs in the polarization of chemotacting cells, and we discuss the possibility that a similar molecular principle may underlie PCP.