Role of a versatile peptide motif controlling Hox nuclear export and autophagy in the Drosophila fat body.

Hox proteins are major regulators of embryonic development, acting in the nucleus to regulate the expression of their numerous downstream target genes. By analyzing deletion forms of the Drosophila Hox protein Ultrabithorax (Ubx), we identified the presence of an unconventional nuclear export signal (NES) that overlaps with a highly conserved motif originally described as mediating the interaction with the PBC proteins, a generic and crucial class of Hox transcriptional cofactors that act in development and cancer. We show that this unconventional NES is involved in the interaction with the major exportin protein CRM1 (also known as Embargoed in flies) in vivo and in vitro We find that this interaction is tightly regulated in the Drosophila fat body to control the autophagy-repressive activity of Ubx during larval development. The role of the PBC interaction motif as part of an unconventional NES was also uncovered in other Drosophila and human Hox proteins, highlighting the evolutionary conservation of this novel function. Together, our results reveal the extreme molecular versatility of a unique short peptide motif for controlling the context-dependent activity of Hox proteins both at transcriptional and non-transcriptional levels.

Broad maternal geographic origin of domestic sheep in Anatolia and the Zagros.

We investigated the controversial origin of domestic sheep (Ovis aries) using large samples of contemporary and ancient domestic individuals and their closest wild relatives: the Asiatic mouflon (Ovis gmelini), the urial (Ovis vignei) and the argali (Ovis ammon). A phylogeny based on mitochondrial DNA, including 213 new cytochrome-b sequences of wild Ovism confirmed that O. gmelini is the maternal ancestor of sheep and precluded mtDNA contributions from O. vignei (and O. gmelini × O. vignei hybrids) to domestic lineages. We also produced 54 new control region sequences showing shared haplogroups (A, B, C and E) between domestic sheep and wild O. gmelini which localized the domestication center in eastern Anatolia and central Zagros, excluding regions further east where exclusively wild haplogroups were found. This overlaps with the geographic distribution of O. gmelini gmelini, further suggesting that the maternal origin of domestic sheep derives from this subspecies. Additionally, we produced 57 new CR sequences of Neolithic sheep remains from a large area covering Anatolia to Europe, showing the early presence of at least three mitochondrial haplogroups (A, B and D) in Western colonization routes. This confirmed that sheep domestication was a large-scale process that captured diverse maternal lineages (haplogroups).

Developmental Robustness: The Haltere Case in Drosophila.

Developmental processes have to be robust but also flexible enough to respond to genetic and environmental variations. Different mechanisms have been described to explain the apparent antagonistic nature of developmental robustness and plasticity. Here, we present a "self-sufficient" molecular model to explain the development of a particular flight organ that is under the control of the Hox gene Ultrabithorax (Ubx) in the fruit fly Drosophila melanogaster. Our model is based on a candidate RNAi screen and additional genetic analyses that all converge to an autonomous and cofactor-independent mode of action for Ubx. We postulate that this self-sufficient molecular mechanism is possible due to an unusually high expression level of the Hox protein. We propose that high dosage could constitute a so far poorly investigated molecular strategy for allowing Hox proteins to both innovate and stabilize new forms during evolution.

Hox dosage contributes to flight appendage morphology in Drosophila.

Flying insects have invaded all the aerial space on Earth and this astonishing radiation could not have been possible without a remarkable morphological diversification of their flight appendages. Here, we show that characteristic spatial expression profiles and levels of the Hox genes Antennapedia (Antp) and Ultrabithorax (Ubx) underlie the formation of two different flight organs in the fruit fly Drosophila melanogaster. We further demonstrate that flight appendage morphology is dependent on specific Hox doses. Interestingly, we find that wing morphology from evolutionary distant four-winged insect species is also associated with a differential expression of Antp and Ubx. We propose that variation in the spatial expression profile and dosage of Hox proteins is a major determinant of flight appendage diversification in Drosophila and possibly in other insect species during evolution.

Neofunctionalization in vertebrates: the example of retinoic acid receptors.

Understanding the role of gene duplications in establishing vertebrate innovations is one of the main challenges of Evo-Devo (evolution of development) studies. Data on evolutionary changes in gene expression (i.e., evolution of transcription factor-cis-regulatory elements relationships) tell only part of the story; protein function, best studied by biochemical and functional assays, can also change. In this study, we have investigated how gene duplication has affected both the expression and the ligand-binding specificity of retinoic acid receptors (RARs), which play a major role in chordate embryonic development. Mammals have three paralogous RAR genes--RAR alpha, beta, and gamma--which resulted from genome duplications at the origin of vertebrates. By using pharmacological ligands selective for specific paralogues, we have studied the ligand-binding capacities of RARs from diverse chordates species. We have found that RAR beta-like binding selectivity is a synapomorphy of all chordate RARs, including a reconstructed synthetic RAR representing the receptor present in the ancestor of chordates. Moreover, comparison of expression patterns of the cephalochordate amphioxus and the vertebrates suggests that, of all the RARs, RAR beta expression has remained most similar to that of the ancestral RAR. On the basis of these results together, we suggest that while RAR beta kept the ancestral RAR role, RAR alpha and RAR gamma diverged both in ligand-binding capacity and in expression patterns. We thus suggest that neofunctionalization occurred at both the expression and the functional levels to shape RAR roles during development in vertebrates.

Human HOX Proteins Use Diverse and Context-Dependent Motifs to Interact with TALE Class Cofactors.

HOX proteins achieve numerous functions by interacting with the TALE class PBX and MEIS cofactors. In contrast to this established partnership in development and disease, how HOX proteins could interact with PBX and MEIS remains unclear. Here, we present a systematic analysis of HOX/PBX/MEIS interaction properties, scanning all paralog groups with human and mouse HOX proteins in vitro and in live cells. We demonstrate that a previously characterized HOX protein motif known to be critical for HOX-PBX interactions becomes dispensable in the presence of MEIS in all except the two most anterior paralog groups. We further identify paralog-specific TALE-binding sites that are used in a highly context-dependent manner. One of these binding sites is involved in the proliferative activity of HOXA7 in breast cancer cells. Together these findings reveal an extraordinary level of interaction flexibility between HOX proteins and their major class of developmental cofactors.

Generation of a versatile BiFC ORFeome library for analyzing protein-protein interactions in live Drosophila.

Transcription factors achieve specificity by establishing intricate interaction networks that will change depending on the cell context. Capturing these interactions in live condition is however a challenging issue that requires sensitive and non-invasive methods.We present a set of fly lines, called 'multicolor BiFC library', which covers most of the Drosophila transcription factors for performing Bimolecular Fluorescence Complementation (BiFC). The multicolor BiFC library can be used to probe two different binary interactions simultaneously and is compatible for large-scale interaction screens. The library can also be coupled with established Drosophila genetic resources to analyze interactions in the developmentally relevant expression domain of each protein partner. We provide proof of principle experiments of these various applications, using Hox proteins in the live Drosophila embryo as a case study. Overall this novel collection of ready-to-use fly lines constitutes an unprecedented genetic toolbox for the identification and analysis of protein-protein interactions in vivo.

Genomic and archaeological evidence suggest a dual origin of domestic dogs.

The geographic and temporal origins of dogs remain controversial. We generated genetic sequences from 59 ancient dogs and a complete (28x) genome of a late Neolithic dog (dated to ~4800 calendar years before the present) from Ireland. Our analyses revealed a deep split separating modern East Asian and Western Eurasian dogs. Surprisingly, the date of this divergence (~14,000 to 6400 years ago) occurs commensurate with, or several millennia after, the first appearance of dogs in Europe and East Asia. Additional analyses of ancient and modern mitochondrial DNA revealed a sharp discontinuity in haplotype frequencies in Europe. Combined, these results suggest that dogs may have been domesticated independently in Eastern and Western Eurasia from distinct wolf populations. East Eurasian dogs were then possibly transported to Europe with people, where they partially replaced European Paleolithic dogs.

Inhibitory activities of short linear motifs underlie Hox interactome specificity in vivo.

Hox proteins are well-established developmental regulators that coordinate cell fate and morphogenesis throughout embryogenesis. In contrast, our knowledge of their specific molecular modes of action is limited to the interaction with few cofactors. Here, we show that Hox proteins are able to interact with a wide range of transcription factors in the live Drosophila embryo. In this context, specificity relies on a versatile usage of conserved short linear motifs (SLiMs), which, surprisingly, often restrains the interaction potential of Hox proteins. This novel buffering activity of SLiMs was observed in different tissues and found in Hox proteins from cnidarian to mouse species. Although these interactions remain to be analysed in the context of endogenous Hox regulatory activities, our observations challenge the traditional role assigned to SLiMs and provide an alternative concept to explain how Hox interactome specificity could be achieved during the embryonic development.

The axolotl (Ambystoma mexicanum), a neotenic amphibian, expresses functional thyroid hormone receptors.

Neotenic amphibians such as the axolotl (Ambystoma mexicanum) are often unable to undergo metamorphosis under natural conditions. It is thought that neoteny represents a deviation from the standard course of amphibian ontogeny, affecting the thyroid axis at different levels from the central nervous system to peripheral organs. Thyroid hormone receptors (TRs) that bind the thyroid hormone (TH) T(3) have been described in axolotl. However, the full sequences of TR were needed to better characterize the TH response and to be able to assess their functional capacity at the molecular level. We report that each of the alpha and beta axolotl TRs bind both DNA and TH, and they activate transcription in response to TH in a mammalian cell-based transient transfection assay. Moreover, both TRs are expressed in axolotl tissues. Interestingly, each TR gene generates alternatively spliced isoforms, harboring partial or total deletions of the ligand-binding domain, which are expressed in vivo. Further, we found that in the axolotl, TH regulates the expression of stromelysin 3 and collagenase 3, which are TH target genes in Xenopus. Taken together, these results suggest that axolotl TRs are functional and that the molecular basis of neoteny in the axolotl is not linked to a major defect in TH response in peripheral tissues.

Bimolecular fluorescence complementation (BiFC) in live Drosophila embryos.

Bimolecular fluorescence complementation (BiFC) is a powerful method for studying protein-protein interactions in different cell types and organisms. This method was recently developed in the fruit fly Drosophila melanogaster, allowing analyzing protein interaction properties in a physiologically relevant developing context. Here we present a detailed protocol for performing BiFC with the Venus fluorescent protein in live Drosophila embryos, taking the Hox-PBC partnership as an illustrative test case. This protocol applies to any transcription factor and split fluorescent protein in general.

Molecular insights into the origin of the Hox-TALE patterning system.

Despite tremendous body form diversity in nature, bilaterian animals share common sets of developmental genes that display conserved expression patterns in the embryo. Among them are the Hox genes, which define different identities along the anterior-posterior axis. Hox proteins exert their function by interaction with TALE transcription factors. Hox and TALE members are also present in some but not all non-bilaterian phyla, raising the question of how Hox-TALE interactions evolved to provide positional information. By using proteins from unicellular and multicellular lineages, we showed that these networks emerged from an ancestral generic motif present in Hox and other related protein families. Interestingly, Hox-TALE networks experienced additional and extensive molecular innovations that were likely crucial for differentiating Hox functions along body plans. Together our results highlight how homeobox gene families evolved during eukaryote evolution to eventually constitute a major patterning system in Eumetazoans. DOI: http://dx.doi.org/10.7554/eLife.01939.001.

Evidence of coat color variation sheds new light on ancient canids.

We have used a paleogenetics approach to investigate the genetic landscape of coat color variation in ancient Eurasian dog and wolf populations. We amplified DNA fragments of two genes controlling coat color, Mc1r (Melanocortin 1 Receptor) and CBD103 (canine-ß-defensin), in respectively 15 and 19 ancient canids (dogs and wolf morphotypes) from 14 different archeological sites, throughout Asia and Europe spanning from ca. 12 000 B.P. (end of Upper Palaeolithic) to ca. 4000 B.P. (Bronze Age). We provide evidence of a new variant (R301C) of the Melanocortin 1 receptor (Mc1r) and highlight the presence of the beta-defensin melanistic mutation (CDB103-K locus) on ancient DNA from dog-and wolf-morphotype specimens. We show that the dominant K(B) allele (CBD103), which causes melanism, and R301C (Mc1r), the variant that may cause light hair color, are present as early as the beginning of the Holocene, over 10,000 years ago. These results underline the genetic diversity of prehistoric dogs. This diversity may have partly stemmed not only from the wolf gene pool captured by domestication but also from mutations very likely linked to the relaxation of natural selection pressure occurring in-line with this process.

A dig into the past mitochondrial diversity of Corsican goats reveals the influence of secular herding practices.

The goat (Capra hircus) is one of the earliest domesticated species ca. 10,500 years ago in the Middle-East where its wild ancestor, the bezoar (Capra aegagrus), still occurs. During the Neolithic dispersal, the domestic goat was then introduced in Europe, including the main Mediterranean islands. Islands are interesting models as they maintain traces of ancient colonization, historical exchanges or of peculiar systems of husbandry. Here, we compare the mitochondrial genetic diversity of both medieval and extant goats in the Island of Corsica that presents an original and ancient model of breeding with free-ranging animals. We amplified a fragment of the Control Region for 21 medieval and 28 current goats. Most of them belonged to the A haplogroup, the most worldwide spread and frequent today, but the C haplogroup is also detected at low frequency in the current population. Present Corsican goats appeared more similar to medieval goats than to other European goat populations. Moreover, 16 out of the 26 haplotypes observed were endemic to Corsica and the inferred demographic history suggests that the population has remained constant since the Middle Ages. Implications of these results on management and conservation of endangered Corsican goats currently decimated by a disease are addressed.

Pedomorphosis revisited: thyroid hormone receptors are functional in Necturus maculosus.

Heterochrony, a difference in developmental timing, is a central concept in modern evolutionary biology. An example is pedomorphosis, retention of juvenile characteristics in sexually mature adults, a phenomenon largely represented in salamanders. The mudpuppy (Necturus maculosus) is an obligate pedomorphic amphibian, never undergoing metamorphosis. Thyroid hormone induces tissue transformation in metamorphosing species and this action is mediated by nuclear thyroid hormone (TH) receptors (TRs). The absence of metamorphosis in Necturus has been attributed to a resistance to TH action as treatment with exogenous TH fails to induce transformation. The failure to metamorphose could be due to the lack of TR expression in target tissues, or to a loss of TR function. Toward understanding the molecular basis for the failure of Necturus tissues to respond to TH, and the ultimate cause for the expression of the obligate pedomorphic life history, we characterized the structure, function, and expression of TR genes in Necturus. Strikingly, we found that Necturus TRalpha and TRbeta genes encode fully functional TR proteins. These TRs bind both DNA and TH and can transactivate target genes in response to TH. Both TRalpha and TRbeta are expressed in various tissues. TH treatment in vivo induced expression in the gill of some but not all genes known to be activated by TH in anuran larvae, caused whole organism metabolic effects, but induced no external morphological changes in adults or larvae. Thus, Necturus possesses fully functional TRs and its tissues are not generally resistant to the actions of TH. Rather, the absence of metamorphosis may be due to the loss of TH-dependent control of key genes required for tissue transformation.

Molecular cloning and developmental expression patterns of thyroid hormone receptors and T3 target genes in the turbot (Scophtalmus maximus) during post-embryonic development.

Thyroid hormones (TH) are pleiotropic factors important for many developmental and physiological functions in vertebrates and particularly in amphibian metamorphosis. Their effects are mediated by two specific receptors (TRalpha and TRbeta), which are ligand-dependent transcription factors, members of the nuclear hormone receptor superfamily. Besides their pivotal role in amphibian metamorphosis, TH are also critical for fish metamorphosis. As this later role of TH is less studied, we analyzed their action in the turbot (Scophtalmus maximus), a metamorphosing flat fish. We describe the isolation of sequences for the turbot orthologs of a number of Xenopus genes, which are induced during amphibian metamorphosis. Developmental expression of these genes during turbot metamorphosis was studied by several methods and the expression patterns of these genes compared with those in Xenopus and flounder. We find that the period between the onset and the end of eye migration (day 22 to day 30 post-hatching) most likely corresponds to the metamorphic climax with either high TRalpha or high TH levels. Our results show that in contrast to amphibians, it is TRalpha and not TRbeta mRNA that is up-regulated during metamorphosis. Our results highlight the notion that TH regulates, through a rise of TR expression, a genetic cascade during turbot metamorphosis. The fact that TH regulates metamorphosis in amphibian and teleost fishes suggests that TH-regulated metamorphosis is a post-embryonic process conserved in most vertebrates.