Candidate promoter sequences of the solitary genes.

<p>A: detail of the P element showing the sequences used to generate the position-specific weight matrix for homeodomain factor binding sites; on the main sequence, 3'-pointing arrows in salmon show position and orientation of homeodomain factor binding sites as annotated by Vassalli <i>et al</i>.; blue arrows, also 3'-oriented, show emipalindromes identified by EMBOSS Palindrome and used to extend sequences to be added to the matrix; grey boxes mark sequence stretches conserved between the P element and the neighboring <i>Olfr713</i> (<i>P3</i>) promoter (Conserved P-P3) or between the P and the H element (Conserved P-H); a matrix (depicted on the right, top) was obtained aligning the three extended binding sites on P element and the single extended binding site on H element (identical to the central binding site on P element). B: on the putative promoter of <i>Olfr266</i> (3'-pointing grey arrow) a homeodomain factor binding site (3'-pointing salmon arrow labelled as HDBS) is predicted; also, sequences conserved in Mammalia Eutheria are found (green boxes); more towards the 3'-end of the putative promoter, SNPs that are present in the wild for <i>Mus musculus domesticus</i> are found in two positions. C: multi-alignments of candidate promoter sequences (5'-3') from different mouse populations for the solitary genes plus (orange box) the non-solitary, reference odorant receptor gene <i>Olfr6</i> (<i>M50</i>); for <i>Olfr466</i> two different sequences are presented, one 5' RACE-derived (tagged with [R]) and one based on Plessy <i>et al</i>. (labelled with [P]); B6 stands for C57BL/6J, W stands for consensus of all wild-caught populations, F stands for French population, G stands for German population and I stands for Iranian population; in brown are marked mutant C57BL/6J bases; in violet are highlighted bases differing from consensus; thin vertical lines (light blue) on sequence alignment mark the position of variations in at least some of the 36 non-C57BL/6J laboratory strains, reporting also the fraction of strains carrying the variation (above) and the variation details (below); for <i>Olfr266</i>, sequences boxed in green are conserved in Mammalia Eutheria, and bases in arrowed salmon box (arrow points towards 3') are part of the predicted homeodomain factor binding site.</p

5' RACE clones aligned against their genomic references.

<p>For each gene, middle black line represents the genomic <i>locus</i> (GRCm38.p1). Transcription start sites derived from literature are labelled with capital letters (C for Clowney <i>et al</i>., P for Plessy <i>et al</i>., Y for Young <i>et al</i>.). Light blue arrows depict coding sequences as they appear on Ensembl. Fuchsia arrows represent sequences selected as putative promoters. On <i>Olfr466</i>, a pink arrow renders an alternative putative promoter derived from Plessy <i>et al</i>. All arrows point towards 3'-end. Above genomic <i>loci</i>, grey stacked lines represent spliced 5' RACE clones obtained from whole olfactory mucosa; similarly, in green below the genomic reference, clones derived from sorted olfactory sensory neurons are depicted. Thin (grey or green) lines in 5' RACE clones represent introns. Three dots (…) on a stacked line indicate that the clone is not sequenced until its 3'-end. Shortest clones are omitted for graphical reasons. The control, non-solitary, odorant receptor gene <i>Olfr6</i> (<i>M50</i>) is boxed in orange.</p

Solitary genes expressed in the mouse main olfactory epithelium.

<p><i>In situ</i> hybridization of the main olfactory epithelium of three-day-old mice (n = 3), showing the expression of the eight mouse solitary genes: <i>Olfr19</i> (<i>M12</i>), <i>Olfr49</i>, <i>Olfr266</i>, <i>Olfr267</i>, <i>Olfr370</i>, <i>Olfr371</i>, <i>Olfr466</i> and <i>Olfr1402</i> (green). Nuclear counterstaining is with DAPI (magenta). The table on the right reports the average number of olfactory sensory neurons (± a standard deviation) for each solitary gene. Scale bars: 20 μm.</p

Chromosomal distribution of the solitary genes in Muridae.

<p>Solitary genes for <i>Mus musculus</i> (GRCm38.p1) and <i>Rattus norvegicus</i> (RGSC 3.4) are indicated in light green if intact, in dark green if pseudogenized. Grey dashed lines connect each solitary gene with its ortholog. Yellow and orange squares, amplified below the chromosomal diagram of the corresponding species, show pairs of solitary genes that (based on annotation) retain a conserved synteny in Muridae (while a non-annotated, neighboring odorant receptor gene is present for <i>Olr390</i>). The distance (measured in Mb) between genes of each gene pair is annotated. Chromosome bands represent Giemsa staining.</p

Sox10 Expression in Goldfish Retina and Optic Nerve Head in Controls and after the Application of Two Different Lesion Paradigms

<div><p>The mammalian central nervous system (CNS) is unable to regenerate. In contrast, the CNS of fish, including the visual system, is able to regenerate after damage. Moreover, the fish visual system grows continuously throughout the life of the animal, and it is therefore an excellent model to analyze processes of myelination and re-myelination after an injury. Here we analyze Sox10⁺ oligodendrocytes in the goldfish retina and optic nerve in controls and after two kinds of injuries: cryolesion of the peripheral growing zone and crushing of the optic nerve. We also analyze changes in a major component of myelin, myelin basic protein (MBP), as a marker for myelinated axons. Our results show that Sox10⁺ oligodendrocytes are located in the retinal nerve fiber layer and along the whole length of the optic nerve. MBP was found to occupy a similar location, although its loose appearance in the retina differed from the highly organized MBP⁺ axon bundles in the optic nerve. After optic nerve crushing, the number of Sox10⁺ cells decreased in the crushed area and in the optic nerve head. Consistent with this, myelination was highly reduced in both areas. In contrast, after cryolesion we did not find changes in the Sox10⁺ population, although we did detect some MBP^- degenerating areas. We show that these modifications in Sox10⁺ oligodendrocytes are consistent with their role in oligodendrocyte identity, maintenance and survival, and we propose the optic nerve head as an excellent area for research aimed at better understanding of de- and remyelination processes.</p></div

Sox10⁺ cells and MBP⁺ axons in the ONH after PGZ cryolesion.

<p>Cells were counterstained with DAPI. <b>(A)</b> Statistical analysis of Sox10⁺ cell density in the ONH after PGZ cryolesion. No significant differences were found. Data are shown as means± standard deviations. n = 4 animals per group. <b>(B-D)</b> Sox10⁺ cells from 7d, 15d and 30d after injury. <b>(E-G)</b> MBP⁺ axons with degenerating areas (arrows) and growing Zn8⁺ axons (empty arrows) from 7d, 15d and 60d. CA: central artery. Scale bars: B-G: 100 μm.</p

Ultrastructural study of ONH after ON crushing.

<p><b>(A)</b> Areas in degeneration (empty arrows) and the area analyzed in B (asterisk). <b>(B)</b> The micrograph shows areas in degeneration (empty arrows), a large number of unmyelinated axons (Ax) and few myelinated axons (empty arrowheads). Microglia cells (Mic) associated with Müller end feet (Mü) are detected. <b>(C)</b> Oligodendrocyte (Olig). Square enlarged in D. <b>(D)</b> Microtubules (m). <b>(E)</b> Area analyzed in F (asterisk). <b>(F)</b> The micrograph shows a large number of unmyelinated axons (Ax). Numerous microglia cells (Mic), blood cells (Bl) and astrocyte processes with intermediate filaments (f) are seen, as well as the cytoplasm of some immature cells (Im). BL: basal lamina, CA: central artery, f: intermediate filaments, mit: mitochondria, Nuc: nucleolus, desmosomes (arrowheads). Scale bars: A, E: 50 μm; B, F: 2500 nm; C-D: 1000 nm.</p

Primers used in PCR assays.

<p>Primers used in PCR assays.</p

Sox10 time-course analysis after ON crushing.

<p><b>(A)</b> ONH Sox10⁺cell counts in sections. <b>(B)</b> Retinal Sox10⁺ cells in whole-mount retinas. **p<0.01. Data are shown as means± standard deviations. n = 4 animals per group.</p

Antibodies used in the present work.

<p>Antibodies used in the present work.</p