Walter Jakob Gehring

Quantitative confocal fluorescence microscopy imaging without scanning is developed for the study of fast dynamical processes. The method relies on the use of massively parallel Fluorescence Correlation Spectroscopy (mpFCS). Simultaneous excitation of fluorescent molecules across the specimen is achieved by passing a single laser beam through a Diffractive Optical Element (DOE) to generate a quadratic illumination matrix of 32×32 light sources. Fluorescence from 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector consisting of the same number of single-photon avalanche photodiodes (SPADs). Software was developed for data acquisition and fast autoand cross-correlation analysis by parallel signal processing using a Graphic Processing Unit (GPU). Instrumental performance was assessed using a conventional single-beam FCS instrument as a reference. Versatility of the approach for application in biomedical research was evaluated using ex vivo salivary glands from Drosophila third instar larvae expressing a fluorescently-tagged transcription factor Sex Combs Reduced (Scr) and live PC12 cells stably expressing the fluorescently tagged mu-opioid receptor (MOP_eGFP). We show that quantitative mapping of local concentration and mobility of transcription factor molecules across the specimen can be achieved using this approach, which paves the way for future quantitative characterization of dynamical reaction-diffusion landscapes across live cells/tissue with a submillisecond temporal resolution (presently 21 μs/frame) and single-molecule sensitivity.

In this review, the evolution of vision is retraced from its putative origins in cyanobacteria to humans. Circadian oscillatory clocks, phototropism, and phototaxis require the capability to detect light. Photosensory proteins allow us to reconstruct molecular phylogenetic trees. The evolution of animal eyes leading from an ancestral prototype to highly complex image forming eyes can be deciphered on the basis of evolutionary developmental genetic experiments and comparative genomics. As all bilaterian animals share the same master control gene, Pax6, and the same retinal and pigment cell determination genes, we conclude that the different eye‐types originated monophyletically and subsequently diversified by divergent, parallel, or convergent evolution. WIREs Dev Biol 2014, 3:1–40. doi: 10.1002/wdev.96This article is categorized under: Gene Expression and Transcriptional Hierarchies > Regulatory Mechanisms Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics Early Embryonic Development > Development to the Basic Body Plan Comparative Development and Evolution > Organ System Comparisons Between Species

Eyes absent (Eya) is a member of the Retinal Determination Gene Network (RDGN), a set of genes responsible for eye specification in Drosophila. Eya is a dual function protein, working as a transcription factor in the nucleus and as a tyrosine phosphatase in the cytoplasm. It had been shown that Pax and Six family genes, main components of the RDGN, are present in the hydrozoan Cladonema radiatum and that they are expressed in the eye. However, nothing had been known about the Eya family in hydrozoan jellyfish. Here we report the presence of an Eya homologue (CrEya) in Cladonema. Real‐time PCR analysis and in situ hybridization showed that CrEya is expressed in the eye. Furthermore, the comprehensive survey of eukaryote genomes revealed that the acquisition of the N‐terminal transactivation domain, including the EYA Domain 2 and its adjacent sequence shared by all eumetazoans, happened early in evolution, before the separation of Cnidaria and Bilateria. Our results uncover the evolution of the two domains and show a conservation of the expression pattern of the Eya gene between Cnidaria and Bilateria, which, together with previous data, supports the hypothesis of the monophyletic origin of metazoans eyes. We additionally show that CrEya is also expressed in the oocytes, where two other members of the RDGN, CrPaxB, and Six4/5‐Cr, are known to be expressed. These data suggest that several members of the RDGN have begun to be localized also into the different context of egg development early in the course of metazoan evolution. J. Exp. Zool. (Mol. Dev. Evol.) 318:257–267, 2012.

Developmental genetics of Drosophila continue to have a great impact on our understanding of evolution. The specification of the body plan involves four conceptual steps: 1) Localization of maternal mRNAs in the egg cytoplasm. 2) Translation of these RNAs and formation of morphogen gradients. 3) Subdivision of the antero-posterior gradient into a repetitive pattern of body segments. 4) Assignment of a specific identity to each segment by the Hox genes. The discovery of the Hox genes has uncovered a universal principle shared by all bilaterians; they serve as master control genes specifying organization along the antero-posterior axis. The ancestral arthropods presumably consisted of a series of more or less identical segments, which may be represented by recently discovered precambrian Lobopodia which have a pair of legs and a pair of eyes in each segment. The progressive divergence of Hox genes has led to progressive cephalization and caudalization. From the amino acid sequences of the clustered homeodomains we can deduce that the mesothoracic segment represents the prototype from the more anterior and the more posterior segments evolved. Pax6 has been identified as a master control gene for eye development in all bilaterians. Since Pax6 is involved in eye development in all bilaterian phyla, this argues strongly for a monophyletic origin of the metazoan eye. With the same tool box of transcription factors all the different eye-types can be constructed.

Homeotic mutations first found in Drosophila led to the identification of Hox genes in all bilateria. These genes are exceptional in that they are arranged in an ordered cluster, in which they are positioned in the same order along the chromosome as they are expressed along the antero-posterior axis to specify the corresponding body regions. They share a highly conserved DNA sequence of 180 bp, the homeobox which encodes the homeodomain, a 60 amino acid polypeptide involved in specific DNA and RNA binding and in protein-protein interactions. The discovery of the homeobox has uncovered for the first time a universal principle of specification of the body plan along the antero-posterior axis. The structure of the homeodomain has been determined by NMR spectroscopy and by X-ray crystallography. However, the mechanism by which the Hox proteins find their target genes in the nucleus of a living cell has been enigmatic. Transcriptome analysis indicates that there are hundreds of target genes to be regulated, both positively and negatively to ensure normal development. In the following, we show by Fluorescence Correlation Spectroscopy (FCS) and single molecule imaging in live salivary gland cells, that the mechanism of recognition is purely stochastic. The homeodomain associates and dissociates rapidly (in the ms range) with chromatin all along the chromosomes. If, however, it associates with a specific binding site in a puffed chromosome region, it remains bound for seconds or minutes to exert its function, by forming a complex with co-activators or co-repressors respectively. These direct measurements solve an old enigma of how Hox transcription factors find their target genes in the nucleus of live cells.

Segmental identity along the anteroposterior axis of bilateral animals is specified by Hox genes. These genes encode transcription factors, harboring the conserved homeodomain and, generally, a YPWM motif, which binds Hox cofactors and increases Hox transcriptional specificity in vivo. Here we derive synthetic Drosophila Antennapedia genes, consisting only of the YPWM motif and homeodomain, and investigate their functional role throughout development. Synthetic peptides and full-length Antennapedia proteins cause head-to-thorax transformations in the embryo, as well as antenna-to-tarsus and eye-to-wing transformations in the adult, thus converting the entire head to a mesothorax. This conversion is achieved by repression of genes required for head and antennal development and ectopic activation of genes promoting thoracic and tarsal fates, respectively. Synthetic Antennapedia peptides bind DNA specifically and interact with Extradenticle and Bric-a-brac interacting protein 2 cofactors in vitro and ex vivo. Substitution of the YPWM motif by alanines abolishes Antennapedia homeotic function, whereas substitution of YPWM by the WRPW repressor motif, which binds the transcriptional corepressor Groucho, allows all proteins to act as repressors only. Finally, naturally occurring variations in the size of the linker between the homeodomain and YPWM motif enhance Antennapedia repressive or activating efficiency, emphasizing the importance of linker size, rather than sequence, for specificity. Our results clearly show that synthetic Antennapedia genes are functional in vivo and therefore provide powerful tools for synthetic biology. Moreover, the YPWM motif is necessary-whereas the entire N terminus of the protein is dispensable-for Antennapedia homeotic function, indicating its dual role in transcriptional activation and repression by recruiting either coactivators or corepressors.

Charles Darwin has proposed the theory that evolution of live organisms is based on random variation and natural selection. Jacques Monod in his classic book Chance and Necessity, published 40 years ago, presented his thesis “that the biosphere does not contain a predictable class of objects or events, but constitutes a particular occurrence, compatible indeed with the first principles, but not deducible from those principals and therefore, essentially unpredictable.” Recent discoveries in eye evolution are in agreement with both of these theses. They confirm Darwin's assumption of a simple eye prototype and lend strong support for the notion of a monophyletic origin of the various eye types. Considering the complexity of the underlying gene regulatory networks the unpredictability is obvious. The evolution of the Hox gene cluster and the specification of the body plan starting from an evolutionary prototype segment is discussed. In the course of evolution, a series of similar prototypic segments gradually undergoes cephalization anteriorly and caudalization posteriorly through diversification of the Hox genes.

Pax transcription factors are involved in a variety of developmental processes in bilaterians, including eye development, a role typically assigned to Pax-6. Although no true Pax-6 gene has been found in nonbilateral animals, some jellyfish have eyes with complex structures. In the cubozoan jellyfish Tripedalia , Pax-B , an ortholog of vertebrate Pax-2/5 / 8 , had been proposed as a regulator of eye development. Here we have isolated three Pax genes ( Pax-A , Pax-B , and Pax-E ) from Cladonema radiatum , a hydrozoan jellyfish with elaborate eyes. Cladonema Pax-A is strongly expressed in the retina, whereas Pax-B and Pax-E are highly expressed in the manubrium, the feeding and reproductive organ. Misexpression of Cladonema Pax-A induces ectopic eyes in Drosophila imaginal discs, whereas Pax-B and Pax-E do not. Furthermore, Cladonema Pax-A paired domain protein directly binds to the 5′ upstream region of eye-specific Cladonema opsin genes, whereas Pax-B does not. Our data suggest that Pax-A , but not Pax-B or Pax-E , is involved in eye development and/or maintenance in Cladonema . Phylogenetic analysis indicates that Pax-6 , Pax-B , and Pax-A belong to different Pax subfamilies, which diverged at the latest before the Cnidaria–Bilateria separation. We argue that our data, showing the involvement of Pax genes in hydrozoan eye development as in bilaterians, supports the monophyletic evolutionary origin of all animal eyes. We then propose that during the early evolution of animals, distinct classes of Pax genes, which may have played redundant roles at that time, were flexibly deployed for eye development in different animal lineages.

In The Origin of Species, Darwin proposed that the complex eyes of animals evolved from a simple prototype consisting of two cells only, a photoreceptor cell and a pigment cell shielding the light from one side and allowing for a directional vision. The discovery of Pax6 as a master control gene for eye development in bilateria lends considerable support to the hypothesis of a monophyletic origin of the metazoan eyes from a Darwinian prototype. Pax6 is found in all bilateria and it is capable of inducing ectopic eyes in both vertebrates and invertebrates. The Darwinian prototype eyes have been found in nature in certain planarians and in the trochophora larvae of Platynereis, an annelid worm, where they were found to express Pax6. The prototypic eye must have evolved from a single cell. Such a single-celled pigmented photoreceptor has been found in the larvae of Tripedalia, a jellyfish, whereas the adult has multicellular lens eyes. We propose that the evolution of the prototypic eye started with a first step of cellular differentiation from a single-celled pigmented photoreceptor, into two cell types: a photoreceptor cell specified by Pax6, and a pigment cell specified by the microphthalmia transcription factor (Mitf), which is also conserved from jellyfish to humans. From the prototypic eye more complex eyes can evolve by intercalating additional genes into the eye developmental pathway by gene duplication and subsequent divergence or by enhancer fusion.