Faculty Bibliography

Drosophila has recently become a powerful model system to understand the mechanisms of temporal patterning of neural progenitors called neuroblasts (NBs). Two different temporal sequences of transcription factors (TFs) have been found to be sequentially expressed in NBs of two different systems: the Hunchback, Kruppel, Pdm1/Pdm2, Castor, and Grainyhead sequence in the Drosophila ventral nerve cord; and the Homothorax, Klumpfuss, Eyeless, Sloppy-paired, Dichaete, and Tailless sequence that patterns medulla NBs. In addition, the intermediate neural progenitors of type II NB lineages are patterned by a different sequence: Dichaete, Grainyhead, and Eyeless. These three examples suggest that temporal patterning of neural precursors by sequences of TFs is a common theme to generate neural diversity. Cross-regulations, including negative feedback regulation and positive feedforward regulation among the temporal factors, can facilitate the progression of the sequence. However, there are many remaining questions to understand the mechanism of temporal transitions. The temporal sequence progression is intimately linked to the progressive restriction of NB competence, and eventually determines the end of neurogenesis. Temporal identity has to be integrated with spatial identity information, as well as with the Notch-dependent binary fate choices, in order to generate specific neuron fates.

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s. Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc and initiates the assembly of undifferentiated cells into ommatidia. The 'founder cell' R8 is specified first and recruits R1-6 and then R7. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes, including rhabdomere formation, synaptogenesis and eventually rh expression. In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

There are two views on vertebrate retinogenesis: a deterministic model dependent on fixed lineages and a stochastic model in which choices of division modes and cell fates cannot be predicted. In this issue of Neuron, He et al. (2012) address this question in zebrafish using live imaging and mathematical modeling.

The development of two-component expression systems in Drosophila melanogaster, one of the most powerful genetic models, has allowed the precise manipulation of gene function in specific cell populations. These expression systems, in combination with site-specific recombination approaches, have also led to the development of new methods for clonal lineage analysis. We present a hands-on user guide to the techniques and approaches that have greatly increased resolution of genetic analysis in the fly, with a special focus on their application for lineage analysis. Our intention is to provide guidance and suggestions regarding which genetic tools are most suitable for addressing different developmental questions.

Color vision is found in many invertebrate and vertebrate species. It is the ability to discriminate objects based on the wavelength of emitted light independent of intensity. As it requires the comparison of at least two photoreceptor types with different spectral sensitivities, this process is often mediated by a mosaic made of several photoreceptor types. In this review, we summarize the current knowledge about the formation of retinal mosaics and the regulation of photopigment (opsin) expression in the fly, mouse, and human retina. Despite distinct evolutionary origins, as well as major differences in morphology and phototransduction machineries, there are significant similarities in the stepwise cell-fate decisions that lead from progenitor cells to terminally differentiated photoreceptors that express a particular opsin. Common themes include (i) the use of binary transcriptional switches that distinguish classes of photoreceptors, (ii) the use of gradients of signaling molecules for regional specializations, (iii) stochastic choices that pattern the retina, and (iv) the use of permissive factors with multiple roles in different photoreceptor types.

Patterning the Drosophila retina for color vision relies on postmitotic specification of photoreceptor subtypes. R8 photoreceptors express one of two light-sensing Rhodopsins, Rh5 or Rh6. This fate decision involves a bistable feedback loop between Melted, a PH-domain protein, and Warts, a kinase in the Hippo growth pathway. Here, we show that a subset of the Hippo pathway-Merlin, Kibra, and Lethal(2)giant larvae (Lgl), but not Expanded or Fat-is required for Warts expression and activity in R8 to specify Rh6 fate. Melted represses warts transcription to disrupt Hippo pathway activity and specify Rh5 fate. Therefore, R8 Hippo signaling exhibits ON-or-OFF regulation, promoting mutually exclusive fates. Furthermore, Merlin and Lgl are continuously required to maintain R8 neuronal subtypes. These results reveal roles for Merlin, Kibra, and Lgl in neuronal specification and maintenance and show that the Hippo pathway is reimplemented for sensory neuron fate by combining canonical and noncanonical regulatory steps.

Sensory systems with high discriminatory power use neurons that express only one of several alternative sensory receptor proteins. This exclusive receptor gene expression restricts the sensitivity spectrum of neurons and is coordinated with the choice of their synaptic targets. However, little is known about how it is maintained throughout the life of a neuron. Here we show that the green-light sensing receptor rhodopsin 6 (Rh6) acts to exclude an alternative blue-sensitive rhodopsin 5 (Rh5) from a subset of Drosophila R8 photoreceptor neurons. Loss of Rh6 leads to a gradual expansion of Rh5 expression into all R8 photoreceptors of the ageing adult retina. The Rh6 feedback signal results in repression of the rh5 promoter and can be mimicked by other Drosophila rhodopsins; it is partly dependent on activation of rhodopsin by light, and relies on G(alphaq) activity, but not on the subsequent steps of the phototransduction cascade. Our observations reveal a thus far unappreciated spectral plasticity of R8 photoreceptors, and identify rhodopsin feedback as an exclusion mechanism.

How complex networks of activators and repressors lead to exquisitely specific cell-type determination during development is poorly understood. In the Drosophila eye, expression patterns of Rhodopsins define at least eight functionally distinct though related subtypes of photoreceptors. Here, we describe a role for the transcription factor gene defective proventriculus (dve) as a critical node in the network regulating Rhodopsin expression. dve is a shared component of two opposing, interlocked feedforward loops (FFLs). Orthodenticle and Dve interact in an incoherent FFL to repress Rhodopsin expression throughout the eye. In R7 and R8 photoreceptors, a coherent FFL relieves repression by Dve while activating Rhodopsin expression. Therefore, this network uses repression to restrict and combinatorial activation to induce cell-type-specific expression. Furthermore, Dve levels are finely tuned to yield cell-type- and region-specific repression or activation outcomes. This interlocked FFL motif may be a general mechanism to control terminal cell-fate specification.