The Molecular Structure of cis-4-Aza-A-homo-tetrahydro-a-santonin and trans-4- Aza- A- homo- tetrahydro-a-santonin Related to the Lactam Rule

Stereochemistry of the titled compounds was determined by X-ray structure analysis. The seven-membered lactam rings of both compounds are in a quasi-chair conformation which agrees with the lactam rule. The mean values of the C-NH-CO-C torsion angles are -6 ° and +5°, respectively, and these values also agree with Klyne\u27s hypothesis

Distinct interactions of Sox5 and Sox10 in fate specification of pigment cells in medaka and zebrafish.

Mechanisms generating diverse cell types from multipotent progenitors are fundamental for normal development. Pigment cells are derived from multipotent neural crest cells and their diversity in teleosts provides an excellent model for studying mechanisms controlling fate specification of distinct cell types. Zebrafish have three types of pigment cells (melanocytes, iridophores and xanthophores) while medaka have four (three shared with zebrafish, plus leucophores), raising questions about how conserved mechanisms of fate specification of each pigment cell type are in these fish. We have previously shown that the Sry-related transcription factor Sox10 is crucial for fate specification of pigment cells in zebrafish, and that Sox5 promotes xanthophores and represses leucophores in a shared xanthophore/leucophore progenitor in medaka. Employing TILLING, TALEN and CRISPR/Cas9 technologies, we generated medaka and zebrafish sox5 and sox10 mutants and conducted comparative analyses of their compound mutant phenotypes. We show that specification of all pigment cells, except leucophores, is dependent on Sox10. Loss of Sox5 in Sox10-defective fish partially rescued the formation of all pigment cells in zebrafish, and melanocytes and iridophores in medaka, suggesting that Sox5 represses Sox10-dependent formation of these pigment cells, similar to their interaction in mammalian melanocyte specification. In contrast, in medaka, loss of Sox10 acts cooperatively with Sox5, enhancing both xanthophore reduction and leucophore increase in sox5 mutants. Misexpression of Sox5 in the xanthophore/leucophore progenitors increased xanthophores and reduced leucophores in medaka. Thus, the mode of Sox5 function in xanthophore specification differs between medaka (promoting) and zebrafish (repressing), which is also the case in adult fish. Our findings reveal surprising diversity in even the mode of the interactions between Sox5 and Sox10 governing specification of pigment cell types in medaka and zebrafish, and suggest that this is related to the evolution of a fourth pigment cell type

Comparisons of serum FGF23 (pg/mL).

<p>(<b>A</b>) and Nephron index (<b>B</b>) in chronic kidney disease stages G1, G2, and G3 in diabetic patients. *<i>p</i> < 0.05 vs. G1, **<i>p</i> < 0.01 vs. G1, #<i>p</i> < 0.01 vs. G2, <i>p</i> < 0.01 by post hoc analysis (Holm test) after Kruskal-Wallis test (<i>p</i> < 0.001).</p

Clinical characteristics of the participants with CKD stages G1–G3.

<p>BMI, body mass index; DM, diabetes mellitus; SBP, systolic blood pressure; DBP, diastolic blood pressure; HbA1c, hemoglobin A1c; S-P, serum phosphate; U-P, 24-h urinary excretion of phosphate; S-Ca, serum calcium; 25OH vitD, 25-hydroxyvitamin D3; iPTH, intact parathyroid hormone; eGFR, estimated glomerular filtration rate; U-albumin, urinary albumin.</p

Simple regression analyses of serum P.

<p>(<b>A</b>), Ln intact PTH (<b>B</b>), Ln FGF23 (<b>C</b>), and Ln Nephron index (<b>D</b>) with eGFR in diabetic patients. Pearson’s correlation coefficients were calculated. FGF23, fibroblast growth factor 23; Ln, logarithmic value.</p

Analysis of a novel gene, Sdgc, reveals sex chromosome-dependent differences of medaka germ cells prior to gonad formation

In vertebrates that have been examined to date, the sexual identity of germ cells is determined by the sex of gonadal somatic cells. In the teleost fish medaka, a sex-determination gene on the Y chromosome, DMY/dmrt1bY, is expressed in gonadal somatic cells and regulates the sexual identity of germ cells. Here, we report a novel mechanism by which sex chromosomes cell-autonomously confer sexually different characters upon germ cells prior to gonad formation in a genetically sex-determined species. We have identified a novel gene, Sdgc (sex chromosome-dependent differential expression in germ cells), whose transcripts are highly enriched in early XY germ cells. Chimeric analysis revealed that sexually different expression of Sdgc is controlled in a germ cell-autonomous manner by the number of Y chromosomes. Unexpectedly, DMY/dmrt1bY was expressed in germ cells prior to gonad formation, but knockdown and overexpression of DMY/dmrt1bY did not affect Sdgc expression. We also found that XX and XY germ cells isolated before the onset of DMY/dmrt1bY expression in gonadal somatic cells behaved differently in vitro and were affected by Sdgc. Sdgc maps close to the sex-determination locus, and recombination around the two loci appears to be repressed. Our results provide important insights into the acquisition and plasticity of sexual differences at the cellular level even prior to the developmental stage of sex determination

Distinct interactions of Sox5 and Sox10 in fate specification of pigment cells in medaka and zebrafish - Fig 2

<p><b>Reductions in melanoblast and iridoblast specification in <i>sox10a</i> mutants were partially rescued by loss of <i>sox5</i></b>. (A-D) <i>mitfa</i>. (E-H) <i>dct</i>. (I-L) <i>ltk</i>. (A, E, I) WT. (B, F, J) <i>sox5</i>^<i>-/-</i>. (C, G, K) <i>sox10a</i>^<i>-/-</i>. (D, H, L) <i>sox10a</i>^<i>-/-</i><i>;sox5</i>^<i>-/-</i>. (M-O) Quantitation of number of cells expressing each fate marker in the whole embryonic body (M, N) and in the lateral patches (O). (A-H) Lateral views. (I-L) Dorsal views. (A-H) 30-somite stage (30s, 64 hpf). (I-L) 34-somite stage (34s, 74 hpf). The box in (A) indicates the enlarged region (anterior trunk) in insets. Melanoblasts, defined by expression of <i>mitfa</i> and <i>dct</i>, are unaltered in the <i>sox5</i>^-/- mutant (B, F) as compared with WT (A, E). Expression of <i>mitfa</i> and <i>dct</i> are both dramatically reduced in <i>sox10a</i>^<i>-/-</i> mutants (C, G), but substantially recovered in <i>sox10a</i>^<i>-/-</i><i>;sox5</i>^<i>-/-</i> mutants (D, H). The number of <i>mitfa</i> (M, <i>mitfa</i>^<i>+</i>)- or <i>dct</i> (N, <i>dct</i>^<i>+</i>)- expressing cells is not different between WT (<i>mitfa</i>, n = 9; <i>dct</i>, n = 14) and <i>sox5</i>^<i>-/-</i> mutants (<i>mitfa</i>, n = 15; <i>dct</i>, n = 15). <i>p-</i>values are <i>p</i> = 0.78 (<i>mitfa</i>) and <i>p</i> = 0.52 (<i>dct</i>). <i>sox10a</i>^<i>-/-</i> mutants have significantly fewer of those cells (<i>mitfa</i>, n = 13; <i>dct</i>, n = 12) than WT (<i>p</i><0.05 (m<i>itfa</i>) and <i>p</i><0.05 (<i>dct</i>)). In the <i>sox10a</i>^<i>-/-</i><i>;sox5</i>^<i>-/-</i> double mutants (<i>mitfa</i>, n = 10; <i>dct</i>, n = 13), cell counts are significantly increased as compared with <i>sox10a</i>^<i>-/-</i> single mutant (*, <i>p</i><0.05). <i>p</i>-values were calculated by Mann-Whitney test. Iridoblasts, as evidenced by <i>ltk</i> expression, in WT (I), <i>sox5</i>^<i>-/-</i> mutant (J), <i>sox10a</i>^<i>-/-</i> mutant (K) and <i>sox10a</i>^<i>-/-</i><i>;sox5</i>^<i>-/-</i> mutant (L) are shown for the lateral patches, a region of concentrated iridophores dorsolateral to the developing gut in the anterior trunk. The number of <i>ltk-</i>expressing cells (O, <i>ltk</i>^<i>+</i>) is indistinguishable between WT (I, n = 20) and the <i>sox5</i>^<i>-/-</i> mutant (J, n = 10) (<i>p =</i> 0.98). In the <i>sox10a</i>^<i>-/-</i> mutant (K, n = 18), the number is significantly decreased as compared with WT (I). Again, iridoblasts cell counts between the <i>sox10a</i>^<i>-/-</i> mutant and the <i>sox10a</i>^<i>-/-</i><i>;sox5</i>^<i>-/-</i> mutant (L, n = 23) is statistically different (*, <i>p</i><0.05), showing partial rescue in the double homozygote. <i>p</i>-values were calculated by Mann-Whitney test. (M-O) Bars show mean and error bar (s.d.). Scale bars: (A) 200 μm, (I) 40 μm.</p

Distinct interactions of Sox5 and Sox10 in fate specification of pigment cells in medaka and zebrafish - Fig 6

<p><b>Role of Sox5 in adult xanthophore development in medaka remains opposite to that in zebrafish</b>. (A, B) Medaka. 6 mpf. (D, E) Zebrafish. 2 mpf. (D’, E’) Schematics of stripe pattern. (D”, E”) Enlarged images of X0 interstripe. (F, G) Zebrafish. 1.5 years old. In WT medaka (A), melanocytes, xanthophores (yellow arrowhead) and leucophores (white arrowhead) are scattered on the body surface. <i>sox5</i>^<i>-/-</i> mutant medaka (B) have fewer xanthophores and more leucophores than WT (C, Xan and Leu, *<i>p</i><0.05 by Mann-Whitney test; WT, n = 5; <i>sox5</i>^<i>-/-</i>, n = 5; Bars show mean and error bar (s.d.)). Pigment cells were counted from a 1 mm² area on the dorsal body surface. The melanocyte numbers were not significantly different between WT and <i>sox5</i>^<i>-/-</i> (C, Mel, <i>p</i> = 0.1 by Mann-Whitney test). The adult pigment pattern of WT zebrafish is composed of 5 melanocyte stripes (2D, 1D, 1V, 2V, 3V) and xanthophore interstripes (X1D, X0, X1V, X2V) (D, D’). Zebrafish <i>sox5</i>^<i>-/-</i> mutants lack two ventral interstripes (2V and 3V) (E, E’), and thus have fewer stripes than WT. This is the case regardless of sex (F, female; G, male) after the mutant fish get larger and older than 1.5 years. The <i>sox5</i>^<i>-/-</i> zebrafish mutants have wider X1D and X0 (two-way arrow in D” and E”) interstripes and larger numbers of xanthophores in these interstripes than WT. (H) Scatter plot of stripe width or pigment cell numbers in each stripe, comparing <i>sox5</i>^<i>-/-</i> and their WT siblings. X axis shows the standard body length of zebrafish examined. Analysis of covariance was performed to examine the differences in width or cell numbers between WT and <i>sox5</i>^<i>-/-</i> mutants, by using standard length as a covariate. The <i>p</i> values are as follows; width X1D (<i>p</i><0.05), 1D (<i>p</i><0.05), X0 (<i>p</i><0.05), cell number X1D (<i>p</i><0.05), 1D (<i>p</i> = 0.985), X0 (<i>p</i><0.05). The width and number of xanthophore in the xanthophore stripes (X0, X1D) in <i>sox5</i>^<i>-/-</i> mutants (black boxes) show significant increase compared with WT siblings (white boxes). The number of melanocyte in the 1D stripe is comparable between WT (white boxes) and <i>sox5</i>^<i>-/-</i> mutant (black boxes), but the width is slightly but significantly different (<i>p</i><0.05). Scale bar: (A) 200 μm, (D) 3 mm, (D”) 200 μm, (F) 3 mm.</p

Distinct interactions of Sox5 and Sox10 in fate specification of pigment cells in medaka and zebrafish - Fig 5

<p><b>Sox10-mediated pigment cell formation is modulated by Sox5 in zebrafish</b>. (A, B, C, E) Swim bladder inflation stage (10 dpf). Lateral views. (C, E) UV images. (D, F) 24 dpf. Lateral views. (G, I-O) 4 dpf. (G) Dorsolateral view. (I) Lateral view. (J-O) Fluorescing xanthophores. Dorsal views anterior to the left. The <i>sox5</i> mutant is indistinguishable from WT, exhibiting four stripes of melanocytes (A, B) and having fluorescing xanthophores (C, E) and <i>gch</i>-expressing xanthoblasts (D, F). Melanocytes are almost completely absent from <i>sox10</i>^{<i>baz1/baz1</i>} mutant (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007260#pgen.1007260.s008" target="_blank">S8B Fig</a>), but are partially recovered in <i>sox10</i>^{<i>baz1/baz1</i>} mutants that have also lost <i>sox5</i> WT allele(s) (G, H). The ratio of the embryos without melanocytes or with more than one melanocytes was compared among genotypes by Chi-squared test (*<i>p</i><0.05). A few xanthophores develop on surface of the head in <i>sox10</i>^{<i>baz1/baz1</i>} mutant (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007260#pgen.1007260.s008" target="_blank">S8E, S8I and S8J Fig</a>), and reduction of <i>sox5</i> is likely to elevate xanthophore formation (K, L). Whereas <i>sox10</i>^<i>t3/t3</i> mutant almost completely lacks xanthophores (M), a few xanthophores are rescued as the <i>sox5</i> WT allele(s) are reduced (N, O). The counts are shown for xanthophores on the <i>t3</i> background (P). Comparison between the genotypes was performed by Kruskal-Wallis test with SDCF post hoc test. **<i>p</i><0.05. A substantial number of iridophores are formed in <i>sox10</i>^{<i>baz1/baz1</i>} mutants (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007260#pgen.1007260.s008" target="_blank">S8G and S8H Fig</a>), and the counts are not significantly altered with reduction of the <i>sox5</i> WT allele(s) (Q, <i>p</i> = 0.775 by Kruskal-Wallis test). Iridophores are almost completely lost in <i>sox10</i>^<i>t3/t3</i> mutant (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007260#pgen.1007260.s008" target="_blank">S8I Fig</a>), but are partially recovered in <i>sox10</i>^<i>t3/t3</i><i>;sox5</i>^<i>+/-</i> and <i>sox10</i>^<i>t3/t3</i><i>;sox5</i>^<i>-/-</i> mutants (R **<i>p</i><0.05 by Kruskal-Wallis test with SDCF post hoc test). (H) <i>sox10</i>^{<i>baz1/baz1</i>}<i>;sox5</i>^<i>+/+</i>, n = 22; <i>sox10</i>^{<i>baz1/baz1</i>}<i>;sox5</i>^<i>+/-</i>, n = 34; <i>sox10</i>^{<i>baz1/baz1</i>}<i>;sox5</i>^<i>-/-</i>, n = 33. (Q) <i>sox10</i>^{<i>baz1/baz1</i>}<i>;sox5</i>^<i>+/+</i>, n = 46; <i>sox10</i>^{<i>baz1/baz1</i>}<i>;sox5</i>^<i>+/-</i>, n = 73; <i>sox10</i>^{<i>baz1/baz1</i>}<i>;sox5</i>^<i>-/-</i>, n = 47. (P) <i>sox10</i>^<i>t3/t3</i><i>;sox5</i>^<i>+/+</i>, n = 7; <i>sox10</i>^<i>t3/t3</i><i>;sox5</i>^<i>+/-</i>, n = 19; <i>sox10</i>^<i>t3/t3</i><i>;sox5</i>^<i>-/-</i>, n = 16. (R) <i>sox10</i>^<i>t3/t3</i><i>;sox5</i>^<i>+/+</i>, n = 24; <i>sox10</i>^<i>t3/t3</i><i>;sox5</i>^<i>+/-</i>, n = 48; <i>sox10</i>^<i>t3/t3</i><i>;sox5</i>^<i>-/-</i>, n = 40. (H, P-R) Bars show mean and error bar (s.d.). Arrowheads point to weakly melanised cells (G) and at the corresponding position of the head (I-O). Scale bars: (A, D) 200 μm.</p