Publications

The building of the topographic map in the mammalian olfactory bulb is explained by a model based on two axes along which sensory neurons are guided: one dorso-ventral and the other antero-posterior. This latter axis relies on specific expression levels of Neuropilin 1 (Nrp1). To evaluate the role played by this receptor in this process, we used an in vivo genetic approach to decrease or suppress it in specific neuronal populations and at different time points during axonal targeting. We observed, in neurons that express either the M71 or the M72 odorant receptors, that the inactivation of Nrp1 leads to two distinct wiring alterations, whose incidence depends on the time at which Nrp1 expression is altered: first, a surprising dorsal shift of the M71 and M72 glomeruli that often fuse with their contralateral counterparts, and second, the formation of anteriorized glomeruli. The two phenotypes are partly recapitulated in mice lacking the Nrp1 ligand Semaphorin 3A (Sema3A), and in mice whose sensory neurons express a Nrp1 mutant unable to bind Sema3A. Finally, by using a mosaic conditional approach, we show that M71 axonal fibers can bypass the Nrp1 signals that define their target area, since they are hijacked and coalesce with Nrp1-deficient M71-expressing axons that target somewhere else. Together, these findings show drastically different axonal targeting outcomes dependent on the timing at which Nrp1/Sema3A signaling is altered.

Psammophaga fuegiais a new monothalamid foraminifera discovered in surface sediment samples in the Beagle Channel, South America. The species is a member of the important, globally distributed genus Psammophaga, which has the ability to ingest and store mineral particles inside the cytoplasm. Its shape is ovoid to pyriform, the size varies from 250 to 600 µm in length and from 200 to 400 µm in width. Like other Psammophaga species P. fuegia has a single aperture. It was found in multiple samples across the Beagle Channel area at water depths of 4 to 220 meters and in environments as variable as fjords, the main channel, and the harbour of Puerto Williams (Chile). The occurrences of the new species in environmental DNA and RNA samples correspond well to its distribution inferred from the microscopic study.

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Within land vertebrate species, snakes display extreme variations in their body plan, characterized by the absence of limbs and an elongated morphology. Such a particular interpretation of the basic vertebrate body architecture has often been associated with changes in the function or regulation of Hox genes. Here, we use an interspecies comparative approach to investigate different regulatory aspects at the snake HoxD locus. We report that, unlike in other vertebrates, snake mesoderm-specific enhancers are mostly located within the HoxD cluster itself rather than outside. In addition, despite both the absence of limbs and an altered Hoxd gene regulation in external genitalia, the limb-associated bimodal HoxD chromatin structure is maintained at the snake locus. Finally, we show that snake and mouse orthologous enhancer sequences can display distinct expression specificities. These results show that vertebrate morphological evolution likely involved extensive reorganisation at Hox loci, yet within a generally conserved regulatory framework.

Functionally autonomous regulatory domains direct the parasegment-specific expression of the Drosophila Bithorax complex (BX-C) homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS) regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass) for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the Fab-8 boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp Fab-8 PTS (promoter targeting sequence) fully rescues a Fab-7 deletion. It blocks crosstalk between the iab-6 and iab-7 regulatory domains, and has bypass activity that enables the two downstream domains, iab-5 and iab-6, to regulate Abdominal-B (Abd-B) transcription in spite of two intervening boundary elements. Fab-8 has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw)) sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the Fab-8 replacement. Though the inverted Fab-8 boundary still blocks crosstalk, it disrupts the topology of the Abd-B regulatory domains and does not support bypass. Importantly, altering the orientation of the Fab-8 dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors.

We sequenced Early Neolithic genomes from the Zagros region of Iran (eastern Fertile Crescent), where some of the earliest evidence for farming is found, and identify a previously uncharacterized population that is neither ancestral to the first European farmers nor has contributed substantially to the ancestry of modern Europeans. These people are estimated to have separated from Early Neolithic farmers in Anatolia some 46,000 to 77,000 years ago and show affinities to modern-day Pakistani and Afghan populations, but particularly to Iranian Zoroastrians. We conclude that multiple, genetically differentiated hunter-gatherer populations adopted farming in southwestern Asia, that components of pre-Neolithic population structure were preserved as farming spread into neighboring regions, and that the Zagros region was the cradle of eastward expansion.

Oligochaetes are valuable bioindicators of the quality of watercourse and lake sediments. The morphological identification of aquatic oligochaetes is difficult, prompting the development of new molecular oligochaete indices based on DNA barcoding and Next-generation sequencing of sorted specimens. In general, the samples for DNA barcoding are fixed in absolute ethanol. However, in the case of aquatic oligochaetes, this medium is not appropriate as it can induce a modification of specimen abundances and of the composition of communities. Therefore, we investigated the possibility to amplify and sequence aquatic oligochaetes fixed in formalin for a short time. We performed guanidine extraction and polymerase chain reaction (PCR) amplification/sequencing of the cytochrome c oxydase I (COI) gene on tissue fragments fixed in formalin for different periods of time (from 1 h to 1 week) and in ethanol.

The polymorphism of HLA genes can be used to reconstruct human peopling history. However, this huge diversity impairs successful matching in stem cell transplantation, a situation which has led to the recruitment of millions of donors worldwide. In parallel to the increase of recruitment, registries are progressively relying on information from population genetics to optimize their donor pools in terms of HLA variability. In this study, the HLA data of 65,000 Spanish bone marrow donors were analyzed together with 60,000 Portuguese individuals to provide a comprehensive HLA genetic map of the Iberian Peninsula. The frequencies of many alleles were shown to vary continuously across the Peninsula, either increasing or decreasing from the Mediterranean coast to the Atlantic domain or from the Strait of Gibraltar to the Pyrenees and Bay of Biscay. Similar patterns were observed for several haplotypes. In addition, within some regions neighboring provinces share a close genetic similarity. These results outline the genetic landscape of the Iberian Peninsula, and confirm that the analysis of the HLA polymorphism may reveal relevant signatures of past demographic events even when data from donor registries are used. This conclusion stimulates future developments of the Spanish registry, presented here for the first time.

Most mammals, birds, and reptiles are readily recognized by their hairs, feathers, and scales, respectively. However, the lack of fossil intermediate forms between scales and hairs and substantial differences in their morphogenesis and protein composition have fueled the controversy pertaining to their potential common ancestry for decades. Central to this debate is the apparent lack of an “anatomical placode” (that is, a local epidermal thickening characteristic of feathers’ and hairs’ early morphogenesis) in reptile scale development. Hence, scenarios have been proposed for the independent development of the anatomical placode in birds and mammals and parallel co-option of similar signaling pathways for their morphogenesis. Using histological and molecular techniques on developmental series of crocodiles and snakes, as well as of unique wild-type and EDA (ectodysplasin A)–deficient scaleless mutant lizards, we show for the first time that reptiles, including crocodiles and squamates, develop all the characteristics of an anatomical placode: columnar cells with reduced proliferation rate, as well as canonical spatial expression of placode and underlying dermal molecular markers. These results reveal a new evolutionary scenario where hairs, feathers, and scales of extant species are homologous structures inherited, with modification, from their shared reptilian ancestor’s skin appendages already characterized by an anatomical placode and associated signaling molecules.

In this issue of Developmental Cell, Du et al. (2016) describe a gene named stuxnet that regulates Polycomb protein stability, thereby influencing the activity of the Polycomb-group repressive chromatin complexes.