Masculinity and the Trials of Modern Fiction

This monograph addresses a number of literary trials in nineteenth-century England and France. It examines the ways in which notions of gender were contested and constructed in the literary and legal discourses of the period. It also posits a new model for understanding the complex relationship between law and literature

Increased germ cell apoptosis in <i>fancl</i> mutants at 25 dpf.

<p>Immunodetection of apoptosis by anti-active Caspase-3 in paraffin sections of gonads of wild-type sibling controls (WT) and <i>fancl</i> homozygous mutants (<i>fancl-/-</i>) at 25 dpf (A,B). Presence of Caspase-3-positive cells (shown in red) was lower in gonads of WT (A) than in <i>fancl</i> mutants (B). Gonads outlined by a dashed line (A,B). Bar graph representing the average number of Caspase-3-positive germ cells in each genotype: wild-type sibling controls (WT; n = 6) and <i>fancl</i> homozygous mutants (<i>fancl-/-</i>; n = 6) at 25 dpf (C). Results showed that the average number of apoptotic germ cells in <i>fancl</i> mutants (x– = 99±43) was about three fold higher than in wild-type sibling controls (x– = 35±14), revealing an abnormal increase of germ cell apoptosis in <i>fancl</i> mutants at 25 dpf, a critical period for sex determination (C).</p

Additional file 6: Figure S6. of Glycine-alanine dipeptide repeat protein contributes to toxicity in a zebrafish model of C9orf72 associated neurodegeneration

Tardbp function is not impaired in repeat expressing fish. (A) GA80-GFPa zebrafish expressing GFP and (B) siblings not expressing GFP. Western blot analysis of 2 dpf old embryos with antibodies as indicated. Tardbp/Tardbpl_tv1 bands indicated by arrow heads. (PDF 3086 kb

Zebrafish germ cells express <i>fancl</i> during gonad development.

<p><i>In situ</i> hybridizations with <i>fancl</i> probe were performed on cryo-sections of wild-type animals at different stages of gonad development. Weak <i>fancl</i> expression signal (arrows) was detected in undifferentiated gonads at 17 days post-fertilization (dpf) (A) and 23 dpf (B). Signal became stronger in germ cells (arrows) of transitioning and immature ovaries (ooplasm of oocytes, arrows in C,E,G) and transitioning and immature testes (D,F,H) at 26, 33, and 37 dpf. In adults, <i>fancl</i> expression was restricted to germ cells, but signal intensity depended on the stage of germ cell differentiation. In adult ovaries (I), early stage IB oocytes (<i>e</i>IB) already showed low <i>fancl</i> expression and late stage IB oocytes (<i>l</i>IB) showed strong <i>fancl</i> signal in the ooplasm, suggesting that <i>fancl</i> expression initiated in early stage IB oocytes. As oogenesis progressed, ooplasm volume increased, cortical alveoli appeared (stage II), yolk accumulated (stage III), and <i>fancl</i> expression signal became diluted. In adult testes (J), <i>fancl</i> expression signal was detected in a subset of cells with large nuclei and morphology consistent with primary spermatocytes (sc), but signal was not detected in cells with small nuclei in an advanced stage of spermatogenesis (i.e. spermatids and sperm (sp)). Oocyte staging is according to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001034#pgen.1001034-Selman1" target="_blank">[49]</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001034#pgen.1001034-RodriguezMari1" target="_blank">[29]</a>. Scale bar: 0.1 mm.</p

The absence of females in <i>fancl</i> homozygous mutants is due to sex reversal.

<p>The bar graph represents percentages of expected (ex, grey bars) and observed (ob, black bars) females and males among 211 progeny from a cross of <i>fancl</i> heterozygous females (<i>fancl</i>^+/−) to <i>fancl</i> homozygous mutant males (<i>fancl</i>^−/−). Total numbers (n) and percentages (%) of animals in each category are indicated on the graph. The expected ratio of female heterozygotes to male heterozygotes to female homozygous mutants to male homozygous mutants is 1∶1∶1∶1, but we observed a ratio of about 1∶1∶0∶2 (46 <i>fancl</i>^+/− females: 62 <i>fancl</i>^+/− males: 0 <i>fancl</i>^−/− females: 103 <i>fancl</i>^−/− males). This result rules out the hypothesis that homozygous mutant females die, but is predicted by the hypothesis that homozygous mutants that otherwise would have become females develop instead as males.</p

A model for zebrafish sex determination: oocyte survival regulated by Tp53-mediated apoptosis can alter gonad fate.

<p>This model suggests germ cell apoptosis as a central feature that can integrate genetic and environmental factors to tip the fate of the gonad towards the female or the male pathway and thus determine zebrafish sex. (A) Zebrafish juveniles initially develop an undifferentiated bipotential immature ovary regardless of their eventual definitive sex. The juvenile gonad contains developing oocytes (shown in yellow), as well as somatic cells that express female-specific markers like <i>cyp19a1a</i> (purple) and early male-specific markers like <i>amh</i> (green). This model suggests that different levels of germ cell apoptosis (indicated as a red gradient box from low (white) to high apoptosis (red)) has the potential to tip the fate of the gonad: high apoptosis (e.g <i>fancl^−/−</i> mutants) tips fate towards the male pathway, while low apoptosis (e.g. <i>fancl^−/− tp53^−/−</i> mutants) tips fate towards the female pathway and rescues the sex-reversal phenotype of <i>fancl</i> mutants. In this model, wild-type zebrafish can enter the male pathway at different times during the fate decision time-window (dashed arrows in apoptosis box) (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001034#pgen.1001034-Wang1" target="_blank">[30]</a> and this work), which is probably related to the level of apoptosis that affects oocyte survival in each particular individual. (B) Analysis of somatic markers reported here shows that the survival of oocytes during the fate decision time-window is crucial to maintain and increase expression of <i>cyp19a1a</i> in the somatic cells of the gonad (B, purple gradient) and to down-regulate the expression of <i>amh</i> in the somatic cells of the gonad (B, green gradient), perhaps due to an oocyte-derived signal, which in <i>fancl</i> mutants would be compromised. (C) This gene expression profile feminizes the gonad, oocytes continue to develop, the gonad differentiates as an ovary, and the individual becomes a female. (D) In the absence of oocytes during sex fate decision time, as in <i>fancl</i> mutants, gonads do not maintain <i>cyp19a1a</i> (D, purple gradient), but instead up-regulate <i>amh</i> expression (D, green gradient). (E) This gene expression profile masculinizes the gonad, which differentiates as a mature testis and the individual becomes a male. (F,G) The absence of surviving oocytes in <i>fancl</i>^−/− mutants is probably due to high levels of germ cell apoptosis, which causes all animals to develop as males due to female-to-male sex reversal. This sex reversal phenotype can be rescued by decreasing germ cell apoptosis in double homozygous <i>fancl</i>^−/−;<i>tp53</i>^−/− mutants. Therefore, our analysis of <i>fancl</i> mutants provides evidence supporting the model that the survival of developing oocytes through meiosis, and not the mere presence of germ cells, is a critical factor that tips the fate of the gonad towards the female pathway in zebrafish. (H) Other work has shown that environmental factors such as high temperature can also induce oocyte apoptosis and tip the fate of the gonads towards the male pathway <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001034#pgen.1001034-Uchida2" target="_blank">[71]</a>. In light of this analysis, our model suggests that sex-determining mechanisms in zebrafish integrate signals from genetic and environmental factors that can modify the levels of Tp53-mediated germ cell apoptosis, which influence oocyte survival during the period of gonad fate decision, and tip the fate of the gonad towards the female or the male pathway, thus determining the sex of zebrafish.</p

The Tol2 insertion <i>HG10A</i> disrupts <i>fancl</i> transcripts.

<p>(A) Zebrafish <i>fancl</i> gene structure showing the Tol2 insertion in exon 12 and the position of primer pairs used for RT-PCR experiments (arrows, F1-R1; F2-R2). Numbered boxes represent exons and dashed boxes indicate untranslated regions. (B) Schematic representation from exon 11 to 13 of the wild-type <i>fancl</i> transcript (1.WT) and <i>fancl</i> mutant transcripts (2.<i>fancl</i>Tol2 and 3.<i>fancl</i>D<i>Tol2</i>). The PHD finger domain is highlighted in grey. The Tol2 insertion is shown in black and an arrowhead points to its insertion site in the amino acid sequence in B.2. Predicted protein sequences are shown; the highly conserved Cys and His residues are underlined and the critical Trp is double underlined. Asterisks represent premature stop codons. (C) RT-PCR using as template cDNA of adult testes shows that the 232 bp band containing the intact PHD domain in wild types (amplified by F2-R2 primers) is absent from <i>fancl</i> mutants. The smaller band (174 bp) amplified in <i>fancl</i> mutants corresponds to the <i>fancl</i>D<i>Tol2</i> transcript in B.3. Abbreviations: M, DNA-Marker.</p

Gonads of <i>fancl</i> mutants have germ cells but fail to maintain a female gene expression profile.

<p>Comparative expression analysis of the germ cell marker <i>vasa</i>, the early female somatic cell marker <i>cyp19a1a</i>, and the early male somatic cell marker <i>amh</i> in developing gonads of <i>fancl</i> homozygous mutants (<i>fancl</i>) and their wild-type sibling controls (WT), and in wild-type animals depleted of germ cells by <i>dead end</i> morpholino knockdown (<i>dnd</i>). To monitor the expression of <i>vasa</i>, <i>cyp19a1a</i> and <i>amh</i>, <i>in situ</i> hybridization (ISH) experiments were performed on adjacent cryo-sections of each animal at different stages of gonad development: undifferentiated gonads at 19 dpf (A–I), transitional juvenile gonads at 26 dpf (J–X) and post-transitional juvenile gonads at 33 dpf (Y-M'). Arrows point to examples of regions showing gene expression. ISH with <i>vasa</i> probe labeled germ cells in wild types (A,J,M,Y,B') and <i>fancl</i> mutants (D,P,S,E',H'), and confirmed the depletion of germ cells in <i>dnd</i> animals (G,V,K'). In undifferentiated gonads at 19 dpf, female and male markers were expressed in all genotypes: WT (B,C), <i>fancl</i> (E,F) and <i>dnd</i> knockdown animals (H,I), showing that the onset of <i>cyp19a1a</i> and <i>amh</i> expression does not depend on germ cells or on <i>fancl</i> function. At 26 dpf, controls had started to enter either the male pathway by down-regulating <i>cyp19a1a</i> and up-regulating <i>amh</i> (K,L) or conversely into the female pathway by up-regulating <i>cyp19a1a</i> and down-regulating <i>amh</i> (N,O), correlated with the presence of few or many oocytes, respectively. In contrast, most 26 dpf <i>fancl</i> mutants already showed a male expression profile by the absence of <i>cyp19a1a</i> and the up-regulation of <i>amh</i> (Q,R) and only one <i>fancl</i> mutant showed a low number of <i>cyp19a1a</i>-expressing cells while nevertheless maintaining high <i>amh</i> expression (T,U). Except for <i>vasa</i>, expression profiles of 26 dpf <i>dnd</i> knockdown gonads were similar to <i>fancl</i> mutants (W,X). At 33 dpf, wild-type controls showed either a male expression profile (no <i>cyp19a1a</i> and high <i>amh</i> expression, Z,A') or a female expression profile (high <i>cyp19a1a</i> and no <i>amh</i> expression, C',D'). Most 33 dpf <i>fancl</i> mutants showed a male expression profile (F',G'), even if gonads maintained an ovary-like morphology (I',J'). All 33 dpf <i>dnd</i> animals showed a male expression profile (L',M'). Scale bar: 0.1 mm (A).</p