SM protein Sly1 and a SNARE Habc domain promote membrane fusion through multiple mechanisms.

SM proteins including Sly1 are essential cofactors of SNARE-mediated membrane fusion. Using SNARE and Sly1 mutants and chemically defined in vitro assays, we separate and assess proposed mechanisms through which Sly1 augments fusion: (i) opening the closed conformation of the Qa-SNARE Sed5; (ii) close-range tethering of vesicles to target organelles, mediated by the Sly1-specific regulatory loop; and (iii) nucleation of productive trans-SNARE complexes. We show that all three mechanisms are important and operate in parallel, and that close-range tethering promotes trans-complex assembly when cis-SNARE assembly is a competing process. Further, we demonstrate that the autoinhibitory N-terminal Habc domain of Sed5 has at least two positive activities: it is needed for correct Sed5 localization, and it directly promotes Sly1-dependent fusion. "Split Sed5," with Habc presented solely as a soluble fragment, can function both in vitro and in vivo. Habc appears to facilitate events leading to lipid mixing rather than promoting opening or stability of the fusion pore.

Modulation of tooth regeneration through opposing responses to Wnt and BMP signals in teleosts.

Most vertebrate species undergo tooth replacement throughout adult life. This process is marked by the shedding of existing teeth and the regeneration of tooth organs. However, little is known about the genetic circuitry regulating tooth replacement. Here, we tested whether fish orthologs of genes known to regulate mammalian hair regeneration have effects on tooth replacement. Using two fish species that demonstrate distinct modes of tooth regeneration, threespine stickleback (Gasterosteus aculeatus) and zebrafish (Danio rerio), we found that transgenic overexpression of four different genes changed tooth replacement rates in the direction predicted by a hair regeneration model: Wnt10a and Grem2a increased tooth replacement rate, whereas Bmp6 and Dkk2 strongly inhibited tooth formation. Thus, similar to known roles in hair regeneration, Wnt and BMP signals promote and inhibit regeneration, respectively. Regulation of total tooth number was separable from regulation of replacement rates. RNA sequencing of stickleback dental tissue showed that Bmp6 overexpression resulted in an upregulation of Wnt inhibitors. Together, these data support a model in which different epithelial organs, such as teeth and hair, share genetic circuitry driving organ regeneration.

Distinct tooth regeneration systems deploy a conserved battery of genes.

BACKGROUND: Vertebrate teeth exhibit a wide range of regenerative systems. Many species, including most mammals, reptiles, and amphibians, form replacement teeth at a histologically distinct location called the successional dental lamina, while other species do not employ such a system. Notably, a 'lamina-less' tooth replacement condition is found in a paraphyletic array of ray-finned fishes, such as stickleback, trout, cod, medaka, and bichir. Furthermore, the position, renewal potential, and latency times appear to vary drastically across different vertebrate tooth regeneration systems. The progenitor cells underlying tooth regeneration thus present highly divergent arrangements and potentials. Given the spectrum of regeneration systems present in vertebrates, it is unclear if morphologically divergent tooth regeneration systems deploy an overlapping battery of genes in their naïve dental tissues. RESULTS: In the present work, we aimed to determine whether or not tooth progenitor epithelia could be composed of a conserved cell type between vertebrate dentitions with divergent regeneration systems. To address this question, we compared the pharyngeal tooth regeneration processes in two ray-finned fishes: zebrafish (Danio rerio) and threespine stickleback (Gasterosteus aculeatus). These two teleost species diverged approximately 250 million years ago and demonstrate some stark differences in dental morphology and regeneration. Here, we find that the naïve successional dental lamina in zebrafish expresses a battery of nine genes (bmpr1aa, bmp6, cd34, gli1, igfbp5a, lgr4, lgr6, nfatc1, and pitx2), while active Wnt signaling and Lef1 expression occur during early morphogenesis stages of tooth development. We also find that, despite the absence of a histologically distinct successional dental lamina in stickleback tooth fields, the same battery of nine genes (Bmpr1a, Bmp6, CD34, Gli1, Igfbp5a, Lgr4, Lgr6, Nfatc1, and Pitx2) are expressed in the basalmost endodermal cell layer, which is the region most closely associated with replacement tooth germs. Like zebrafish, stickleback replacement tooth germs additionally express Lef1 and exhibit active Wnt signaling. Thus, two fish systems that either have an organized successional dental lamina (zebrafish) or lack a morphologically distinct successional dental lamina (sticklebacks) deploy similar genetic programs during tooth regeneration. CONCLUSIONS: We propose that the expression domains described here delineate a highly conserved "successional dental epithelium" (SDE). Furthermore, a set of orthologous genes is known to mark hair follicle epithelial stem cells in mice, suggesting that regenerative systems in other epithelial appendages may utilize a related epithelial progenitor cell type, despite the highly derived nature of the resulting functional organs.

The regulation of skin pigmentation in response to environmental light by pineal Type II opsins and skin melanophore melatonin receptors.

Coupling skin colour with the light/dark cycle helps regulate body temperature in ectotherms. In X. laevis, nocturnal release of melatonin from the pineal complex induces pigment aggregation and skin lightening. This nocturnal blanching is initiated by a sensor (type II opsin) that triggers melatonin release when light intensity falls below a minimum threshold, and an effector (melatonin receptor) in the skin which induces pigment aggregation. The sensor/s and effector/s belong to two families of G-protein coupled receptors that originated from a common ancestor, but diverged with subsequent evolution. The aim of this work was to identify candidate sensor/s and effector/s that regulate melatonin-mediated skin colour variation. In X. laevis, we identified a developmental time (stage 43/44) when skin lightening depends on pineal complex photosensitivity alone. At this stage, the pineal complex comprises the frontal organ and pineal gland. A total of 37 type II opsin (14 duplicated) and 6 melatonin receptor (3 duplicated) genes were identified through a full genome analysis of the allotetraploid, X. laevis. These genes were grouped into subfamilies based on their predicted amino acid sequences and the presence of specific amino acids essential for their function. The pineal complex expresses mainly blue light sensitive opsins [pinopsin, parietopsin, opn3, and melanopsins (opn4 and opn4b)] and UV-light sensitive opsins (opn5 and parapinopsin), while visual opsins and va-ancient opsin are absent, as determined by RT-PCR and in situ hybridization. The photoisomerase retinal G-protein coupled receptor, and an uncharacterized opn6b opsin, are also expressed. The spectral sensitivity that triggers melatonin secretion, and therefore melanophore aggregation, falls in the visible spectrum (470-650 Î·m) and peaks in the blue/green range, pointing to the involvement of opsins with sensitivities therein. The effector-melatonin receptors expressed in skin melanophores are mtnr1a and mtnr1c. Our data point to candidate proteins required in the neuroendocrine circuit that underlies the circadian regulation of skin pigmentation, and suggest that multiple initiators and effectors likely participate.

Introgression of regulatory alleles and a missense coding mutation drive plumage pattern diversity in the rock pigeon.

Birds and other vertebrates display stunning variation in pigmentation patterning, yet the genes controlling this diversity remain largely unknown. Rock pigeons (Columba livia) are fundamentally one of four color pattern phenotypes, in decreasing order of melanism: T-check, checker, bar (ancestral), or barless. Using whole-genome scans, we identified NDP as a candidate gene for this variation. Allele-specific expression differences in NDP indicate cis-regulatory divergence between ancestral and melanistic alleles. Sequence comparisons suggest that derived alleles originated in the speckled pigeon (Columba guinea), providing a striking example of introgression. In contrast, barless rock pigeons have an increased incidence of vision defects and, like human families with hereditary blindness, carry start-codon mutations in NDP. In summary, we find that both coding and regulatory variation in the same gene drives wing pattern diversity, and post-domestication introgression supplied potentially advantageous melanistic alleles to feral populations of this ubiquitous urban bird.