Probing the kinetic landscape of Hox transcription factor-DNA binding in live cells by massively parallel Fluorescence Correlation Spectroscopy.

Hox genes encode transcription factors that control the formation of body structures, segment-specifically along the anterior-posterior axis of metazoans. Hox transcription factors bind nuclear DNA pervasively and regulate a plethora of target genes, deploying various molecular mechanisms that depend on the developmental and cellular context. To analyze quantitatively the dynamics of their DNA-binding behavior we have used confocal laser scanning microscopy (CLSM), single-point fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS) and bimolecular fluorescence complementation (BiFC). We show that the Hox transcription factor Sex combs reduced (Scr) forms dimers that strongly associate with its specific fork head binding site (fkh250) in live salivary gland cell nuclei. In contrast, dimers of a constitutively inactive, phospho-mimicking variant of Scr show weak, non-specific DNA-binding. Our studies reveal that nuclear dynamics of Scr is complex, exhibiting a changing landscape of interactions that is difficult to characterize by probing one point at a time. Therefore, we also provide mechanistic evidence using massively parallel FCS (mpFCS). We found that Scr dimers are predominantly formed on the DNA and are equally abundant at the chromosomes and an introduced multimeric fkh250 binding-site, indicating different mobilities, presumably reflecting transient binding with different affinities on the DNA. Our proof-of-principle results emphasize the advantages of mpFCS for quantitative characterization of fast dynamic processes in live cells.

Function and evolutionary origin of unicellular camera-type eye structure.

The ocelloid is an extraordinary eyespot organelle found only in the dinoflagellate family Warnowiaceae. It contains retina- and lens-like structures called the retinal body and the hyalosome. The ocelloid has been an evolutionary enigma because of its remarkable resemblance to the multicellular camera-type eye. To determine if the ocelloid is functionally photoreceptive, we investigated the warnowiid dinoflagellate Erythropsidinium. Here, we show that the morphology of the retinal body changed depending on different illumination conditions and the hyalosome manifests the refractile nature. Identifying a rhodopsin gene fragment in Erythropsidinium ESTs that is expressed in the retinal body by in situ hybridization, we also show that ocelloids are actually light sensitive photoreceptors. The rhodopsin gene identified is most closely related to bacterial rhodopsins. Taken together, we suggest that the ocelloid is an intracellular camera-type eye, which might be originated from endosymbiotic origin.

Evolutionarily conserved transcription factor Apontic controls the G1/S progression by inducing cyclin E during eye development.

During Drosophila eye development, differentiation initiates in the posterior region of the eye disk and progresses anteriorly as a wave marked by the morphogenetic furrow (MF), which demarcates the boundary between anterior undifferentiated cells and posterior differentiated photoreceptors. However, the mechanism underlying the regulation of gene expression immediately before the onset of differentiation remains unclear. Here, we show that Apontic (Apt), which is an evolutionarily conserved transcription factor, is expressed in the differentiating cells posterior to the MF. Moreover, it directly induces the expression of cyclin E and is also required for the G1-to-S phase transition, which is known to be essential for the initiation of cell differentiation at the MF. These observations identify a pathway crucial for eye development, governed by a mechanism in which Cyclin E promotes the G1-to-S phase transition when regulated by Apt.

The evolution of vision.

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.

Cellular uptake of the Antennapedia homeodomain polypeptide by macropinocytosis.

Antennapedia homeodomain has been shown to be able to translocate from extracellular space into the cytoplasm of cells in a receptor-independent manner. Its third α-helix domain, designated as "Penetratin", was proposed to be the functional transduction domain that is responsible for the translocation, and it is widely used for intracellular delivery of various exogenous proteins. Although Penetratin has been regarded to be the only element conferring the capacity on its parent polypeptide to penetrate through the plasma membrane, we found that the complete Antennapedia homeodomain exhibits an appreciably higher level of translocation efficiency as compared to Penetratin. Pharmacological analysis demonstrated that macropinocytic endocytosis plays a significant role underlying the process of the homeodomain internalization, and this is consistent with the observation that internalized polypeptide co-localizes with a fluid phase dye. Our results identify macropinocytosis as a major mechanism by which Antennapedia homeodomain obtains the access to the interior of cells, providing a novel perspective in the field of protein translocation and transduction.

The animal body plan, the prototypic body segment, and eye evolution.

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.

The "eyes absent" (eya) gene in the eye-bearing hydrozoan jellyfish Cladonema radiatum: conservation of the retinal determination network.

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.

Dimer formation via the homeodomain is required for function and specificity of Sex combs reduced in Drosophila.

Hox transcription factors specify body segments along the anteroposterior axis of the embryo. Despite conservation of the homeodomain (HD), different Hox paralogs instruct remarkably different developmental fates. We have unexpectedly found that the Drosophila Sex combs reduced (Scr) protein dimerizes in vivo via the homeodomain, whereas its closest relative, Antennapedia (Antp), does not. Dimerization requires the conserved residue 19 in the ELEKEF motif of the HD and is facilitated by DNA binding. To study Scr dimerization in vivo, we generate a giant transcriptional puff in live salivary gland cells, consisting of a controllable multiple Scr-binding site of the fork head enhancer, and visualize Scr dimer formation upon specific DNA binding. Scr dimerization is required not only for transcriptional activation of the fork head gene but also for Scr homeotic function in the fly (formation of ectopic salivary glands, posterior transformations in the embryo and antenna-to-tarsus transformations). Finally, we attempt to attribute the differential behavior in dimer formation observed between Antp and Scr to diverse amino acid regions between the two proteins that account for dimerization in Scr versus non-dimerization in Antp. By constructing hybrid Antp proteins, we find that the C terminus and linker region between the YPWM motif and the HD of Scr are independently sufficient to confer dimer formation in Antp, whereas the long N terminus of the protein and the HD are largely dispensable. Our results indicate that Scr functions as a homodimer to increase its transcriptional specificity and suggest that the formation of HD homo- or heterodimers might underlie the functional distinction between very similar HD proteins in vivo.

Analyzing the function of a hox gene: an evolutionary approach.

We present an evolutionary approach to dissecting conserved developmental mechanisms. We reason that important mechanisms for making the bodyplan will act early, to generate the major features of the body and that they will be conserved in evolution across many metazoa, and thus, that they will be available in very different animals. This led to our specific approach of microarrays to screen for very early conserved developmental regulators in parallel in an insect, Drosophila and a vertebrate, Xenopus. We screened for the earliest conserved targets of the ectopically expressed hox gene Hoxc6/Antennapedia in both species and followed these targets up, using in situ hybridization, in the Xenopus system. The results indicate that relatively few of the early Hox target genes are conserved: these are mainly involved in the specification of the antero-posterior body axis and in gastrulation.

Chance and necessity in eye evolution.

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.

How do Hox transcription factors find their target genes in the nucleus of living cells?

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.

The ommatidia of Arca noae: a three-tier structure with a central light-guiding element for the receptor cell.

The compound eyes of ark clams appear to function as an optical system to trigger shell closure against predators. We have analyzed the structure of the ommatidia of Arca noae by thin section electron microscopy and serial sectioning, Concanavalin A-gold labeling and acid phosphatase cytochemistry. Our results demonstrate that the ommatidia are a three-tier structure composed of a central single receptor cell, surrounded and covered by proximal pigment cells followed by rows of distal pigment cells. The receptor cells of Arca noae have no lens and the disks of their receptive segment are derived from sensory cilia. The distal mitochondrial segment in the cytoplasm between the nucleus and the receptive segment is surrounded by a mass of Concanavalin A-reactive glycogen particles. Although both, proximal and distal pigment cells have numerous microvilli, only those of the proximal pigment cells form a well-aligned brush border. The microvilli of the latter are ≈9-11 µm long and have a diameter of ≈70-80 nm. Numerous microlamellar bodies cover them. The microlamellar bodies are stored in acid phosphatase-negative secretory granules of the pigment granule-free apical cytoplasm of proximal pigment cells before their secretion. Observation of living compound eyes indicated that the apex of proximal pigment cells transmitted significantly more light than the surrounding distal pigment cells. Hence, the regular geometry of the brush border seems to be a light-guiding structure for receptor cells similar to an optical fiber.

Functional synthetic Antennapedia genes and the dual roles of YPWM motif and linker size in transcriptional activation and repression.

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-à-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.

Flexibly deployed Pax genes in eye development at the early evolution of animals demonstrated by studies on a hydrozoan jellyfish.

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.

Genetic interactions of eyes absent, twin of eyeless and orthodenticle regulate sine oculis expression during ocellar development in Drosophila.

The homeobox gene sine oculis (so) is required for the development of the entire visual system in Drosophila, which includes the compound eyes, the ocelli, the optic lobe of the brain and the Bolwig's organ. During ocelli development, so expression labels, together with eyes absent (eya), the emergence of the ocellar precursor cells in the third instar eye-antennal disc. Footprinting and misexpression studies have led to the proposal that the Pax6 homologue twin of eyeless (toy) directly regulates the initiation of so expression in ocellar precursor cells. However, so expression in a toy loss-of-function mutant background has not been yet analyzed due to the lack of eye-antennal disc development in strong toy mutant alleles. Using an embryonic eye primordium-specific enhancer of toy, we have rescued the developmental defect of a strong toy mutant allele and analyzed so expression in the ocelli primordium of toy loss-of-function eye-antennal discs during third instar larva. The results show that so expression is only marginally affected in the absence of Toy transcriptional activity and that the toy positive effect on so expression is largely eya-mediated. These results suggest that eya is the main factor controlling both initiation and maintenance of so expression in ocellar precursor cells. In addition, we present the characterization of a new minimal eye/ocellus-specific enhancer of the so gene.

Quantitative study of synthetic Hox transcription factor-DNA interactions in live cells.

Transcription factor-DNA interactions are life sustaining and therefore the subject of intensive research. In spite of vast effort, quantitative in vivo studies of the molecular mechanisms underlying these fundamental interactions remain challenging. In the preceding paper, we designed synthetic Sex combs reduced (Scr) peptides and validated genetically their function as transcriptional regulators. Here we present a controllable system for quantitative studies of protein-DNA interactions in live cells that enables us to "titrate" the concentration of the synthetic Scr peptides in a single cell. Using methods with single-molecule sensitivity, advanced fluorescence imaging and fluorescence correlation spectroscopy (FCS), we were able to study the kinetics of Scr-DNA interactions in live salivary gland cells, where Scr is normally expressed during development. We discerned freely moving Scr molecules, characterized the specific and nonspecific Scr peptide-DNA interactions, and estimated their corresponding dissociation constants (K(d)) in vivo. Our results suggest that the synthetic Scr transcription factors find their specific target sites primarily by multiple association/dissociation events, the rapidity of which is largely owed to electrostatic interactions. Based on these new findings, we formulate a model mechanism and emulate the kinetics of Scr homeodomain-DNA interactions in live cells using numerical simulations.

Function and specificity of synthetic Hox transcription factors in vivo.

Homeotic (Hox) genes encode transcription factors that confer segmental identity along the anteroposterior axis of the embryo. However the molecular mechanisms underlying Hox-mediated transcription and the differential requirements for specificity in the regulation of the vast number of Hox-target genes remain ill-defined. Here we show that synthetic Sex combs reduced (Scr) genes that encode the Scr C terminus containing the homedomain (HD) and YPWM motif (Scr-HD) are functional in vivo. Synthetic Scr-HD peptides can induce ectopic salivary glands in the embryo and homeotic transformations in the adult fly, act as transcriptional activators and repressors during development, and participate in protein-protein interactions. Their transformation capacity was found to be enhanced over their full-length counterpart and mutations known to transform the full-length protein into constitutively active or inactive variants behaved accordingly in the synthetic peptides. Our results show that synthetic Scr-HD genes are sufficient for homeotic function in Drosophila and suggest that the N terminus of Scr has a role in transcriptional potency, rather than specificity. We also demonstrate that synthetic peptides behave largely in a predictable way, by exhibiting Scr-specific phenotypes throughout development, which makes them an important tool for synthetic biology.

Evolution of the Hox gene complex from an evolutionary ground state.

In this chapter, we consider the question of how the ordered clusters of Hox genes arose during evolution. Since ordered Hox clusters are found in all major superphyla, we have to assume that the Hox clusters arose before the Cambrian "explosion" giving rise to all of these taxa. Based on his studies of the bithorax complex (BX-C) in Drosophila Lewis considered the ground state to be the mesothoracic segment (T2) since the deletion of all of the genes of the BX-C leads to a transformation of all segments from T3 to A8/9 (the last abdominal segment) into T2 segments. We define the developmental ground state genetically, by assuming that loss-of-function mutants lead to transformations toward the ground state, whereas gain-of-function mutants lead to homeotic transformations away from the ground state. By this definition, T2 also represents the developmental ground state, if one includes the anterior genes, that is, those of the Antennapedia complex. We have reconstructed the evolution of the Hox cluster on the basis of known genetic mechanisms which involve unequal crossover and lead from an urhox gene, first to an anterior and a posterior gene and subsequently to intermediate genes which are progressively inserted, between the anterior and posterior genes. These intermediate genes are recombinant due to unequal crossover, whereas the anterior and posterior genes are not affected and therefore had the longest time to diverge from the urhox gene. The molecular phylogenetic analysis strongly supports this model. We consider the ground state to be both developmental and evolutionary and to represent the prototypic body segment. It corresponds to T2 and is specified by Antennapedia or Hox6, respectively. Experiments in the mouse also suggest that the ground state is a thoracic segment. Evolution leads from the prototypic segment to segmental divergence in both the anterior and posterior direction. The most anterior head and tail segments are specified by homeobox genes localized outside of the cluster.

Spalt major controls the development of the notum and of wing hinge primordia of the Drosophila melanogaster wing imaginal disc.

The Drosophila wing and the dorsal thorax develop from primordia within the wing imaginal disc. Here we show that spalt major (salm) is expressed within the presumptive dorsal body wall primordium early in wing disc development to specify notum and wing hinge tissue. Upon ectopic salm expression, dorsally located second leg disc cells develop notum and wing hinge tissue instead of sternopleural tissue. Similarly, by salm over-expression within the wing disc, wing blade formation is suppressed and a mirror-image duplication of the notum and wing hinge is formed. In large dorsal clones, which lack salm and its neighboring paralogue spalt related (salr), the cells of the notum primordium do not grow; these dorsal cells are not specified as notum, hence no notum outgrowth develops. These results suggest that the zinc finger factors encoded by the salm/salr complex play important roles in defining cells of the early wing disc as dorsal body wall cells, which develop into a large dorsal body wall territory and form mesonotum and some wing hinge tissue, and in delimiting the wing primordium. We also find that salm activity is down-regulated by its own product and by that of the Pax gene eyegone.

Wingless and Hedgehog signaling pathways regulate orthodenticle and eyes absent during ocelli development in Drosophila.

The homeobox gene orthodenticle (otd) controls the process of regional specification that takes place in the Drosophila eye-antennal disc during ocelli development. Mutations that reduce or abolish otd expression in the ocelli primordium give rise to ocelliless flies. We have identified the cis-regulatory sequence (ocelliless enhancer) that controls otd expression during ocelli development and studied its regulation at the molecular level. The ocelliless enhancer is initially activated by the combined action of Wingless (Wg) and Hedgehog (Hh) signaling pathways. Later, a positive autoregulatory feedback loop sets in to maintain otd expression. Moreover, we have analyzed the role of otd during ocelli primordium development and determined its involvement in the expression of the retinal determination gene eyes absent (eya). otd indirectly regulates eya in ocellar precursor cells through the inhibition of wg, an eya repressor, and the maintenance of hh expression in the ocelli primordium. Hh signaling is necessary for eya activation in ocellar precursor cells and this activation is mediated by the full-length activator form of the transcription factor Cubitus interruptus.