Retinoic Acid Metabolic Genes, Meiosis, and Gonadal Sex Differentiation in Zebrafish

<div><p>To help understand the elusive mechanisms of zebrafish sex determination, we studied the genetic machinery regulating production and breakdown of retinoic acid (RA) during the onset of meiosis in gonadogenesis. Results uncovered unexpected mechanistic differences between zebrafish and mammals. Conserved synteny and expression analyses revealed that <i>cyp26a1</i> in zebrafish and its paralog <i>Cyp26b1</i> in tetrapods independently became the primary genes encoding enzymes available for gonadal RA-degradation, showing lineage-specific subfunctionalization of vertebrate genome duplication (VGD) paralogs. Experiments showed that zebrafish express <i>aldh1a2</i>, which encodes an RA-synthesizing enzyme, in the gonad rather than in the mesonephros as in mouse. Germ cells in bipotential gonads of all zebrafish analyzed were labeled by the early meiotic marker <i>sycp3</i>, suggesting that in zebrafish, the onset of meiosis is not sexually dimorphic as it is in mouse and is independent of Stra8, which is required in mouse but was lost in teleosts. Analysis of <i>dead-end</i> knockdown zebrafish depleted of germ cells revealed the germ cell-independent onset and maintenance of gonadal <i>aldh1a2</i> and <i>cyp26a1</i> expression. After meiosis initiated, somatic cell expression of <i>cyp26a1</i> became sexually dimorphic: up-regulated in testes but not ovaries. Meiotic germ cells expressing the synaptonemal complex gene <i>sycp3</i> occupied islands of somatic cells that lacked <i>cyp26a1</i> expression, as predicted by the hypothesis that Cyp26a1 acts as a meiosis-inhibiting factor. Consistent with this hypothesis, females up-regulated <i>cyp26a1</i> in oocytes that entered prophase-I meiotic arrest, and down-regulated <i>cyp26a1</i> in oocytes resuming meiosis. Co-expression of <i>cyp26a1</i> and the pluripotent germ cell stem cell marker <i>pou5f1</i>(<i>oct4</i>) in meiotically arrested oocytes was consistent with roles in mouse to promote germ cell survival and to prevent apoptosis, mechanisms that are central for tipping the sexual fate of gonads towards the female pathway in zebrafish.</p> </div

Complementary expression of the meiotic recombination marker <i>sycp3</i> and <i>cyp26a1</i> in developing gonads.

<p>In bipotential gonads at 20 dpf (A,B: n=8), germ cells expressed the meiotic recombination marker <i>sycp3</i> (black arrowhead in A) in a non-overlapping pattern with <i>cyp26a1</i> expression, which was mostly restricted to the dorsal part of the gonad (revealing that <i>sycp3-</i>expressing cells did not express <i>cyp26a1</i> (red arrowhead in B). Expression of the meiotic marker <i>sycp3</i> was detected in bipotential gonads of all animals analyzed (A, n=8) suggesting that some germ cells entered meiosis in all juveniles regardless of their definitive sex. In differentiating testes at 26 dpf (C,D: n=2) and 29 dpf (G, H: n=2), islands of germ cells that expressed <i>sycp3</i> (black arrowheads in C, G) were found in an area in which RA was likely not degraded due to lack of <i>cyp26a1</i> expression (red arrowheads in D,H). In contrast, in differentiating ovaries at 26 dpf (E, F: n=2) and 29 dpf (I, J: n=2), <i>sycp3</i> was expressed in small germ cells (black arrowheads in E, I) that did not express <i>cyp26a1</i> (red arrowheads in F, J). The expression of <i>cyp26a1</i> was restricted to the ooplasm of oocytes that reached diplotene stage and entered in meiotic arrest (F,J). Scale bar: 0.1mm.</p

Model for the role of retinoic acid and meiotic progression during gonadogenesis in mouse and zebrafish.

<p>In mouse (A), <i>Aldh1a2</i> (yellow) in the mesonephros provides the RA-source that regulates gonad development (reviewed in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073951#B18" target="_blank">18</a>]), while in zebrafish (B), <i>aldh1a2</i> is expressed by somatic cells within the gonad (yellow), and thereby provides an internal RA-source. In mouse, non-meiotic germ cells (black circles) in bipotential gonads are protected from RA by high expression of <i>Cyp26b1</i> (blue), while in zebrafish, Cyp26a1 expression (blue) is restricted to cells at the dorsal surface near the body cavity, and thereby germ cells elsewhere in the gonad are not protected from RA (red) and they are able to enter into meiosis (green circles). In mouse, sexually dimorphic expression of <i>Cyp26b1</i> causes low levels of RA (blue) in testes (males, top in A) and high levels of RA (red) that diffuses from the mesonephros to the ovaries in an anterior to posterior wave (females, bottom in A), which results in a sexually dimorphic onset of meiosis. The onset of meiosis in mouse follows an anterior–posterior wave, accompanied by up-regulation of <i>Stra8</i>, and <i>Sycp3</i> and down-regulation of <i>Pou5f1</i>(Oct4) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073951#B18" target="_blank">18</a>]. In zebrafish, the sexually dimorphic expression of <i>cyp26a1</i> differs in time and location from those of <i>Cyp26b1</i> in mouse. In zebrafish males (top in B), somatic cells up-regulate <i>cyp26a1</i>, and meiotic cells expressing <i>sycp3</i> localize to somatic islands (red) that lack <i>cyp26a1</i> expression. This model is consistent with a role for Cyp26a1 in degrading RA and thereby protecting nearby germ cells from progressing through meiosis. Consistent with this model, in females (bottom in B), <i>cyp26a1</i> is not expressed in somatic cells, but it is up-regulated in the ooplasm (blue) of oocytes at late stage IB that have reached diplotene stage and have entered in meiotic arrest, and also express <i>pou5f1</i>(oct4). The co-expression of <i>cyp26a1</i> and <i>pou5f1</i>(oct4) is compatible with the proposed roles of these genes in mouse on promoting germ cell survival and preventing apoptosis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073951#B41" target="_blank">41</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073951#B86" target="_blank">86</a>], mechanisms that have been shown to be central for tipping the sexual fate of the gonad toward the female pathway in zebrafish [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073951#B5" target="_blank">5</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073951#B8" target="_blank">8</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073951#B87" target="_blank">87</a>].</p

In zebrafish, <i>cyp26a1</i> is the main <i>Cyp26</i> paralog expressed in gonads during the critical period for sex-determination.

<p>Undifferentiated gonads at 15 days post-fertilization (dpf) expressed <i>cyp26a1</i> (A) and <i>cyp26b1</i> (B), but did not express <i>cyp26c1</i> at detectable levels (C) (A-C: n=4). In bipotential gonads at 19 dpf, <i>cyp26a1</i> expression became restricted mainly to the dorsal margin of the gonad (D) but expression of <i>cyp26b1</i> and <i>cyp26c1</i> was not detected (E, F) (D–F: n=9). In differentiating testes at 31 dpf, <i>cyp26a1</i> expression up-regulated (G) and in contrast to mouse testes, which up-regulate <i>cyp26b1</i>, neither <i>cyp26b1</i> nor <i>cyp26c1</i> expression was detected in maturing zebrafish gonads (H, I) (G-I: n=10). Differentiating testes were assigned by morphological features and assessed by the expression of the male specific <i>Amh</i> marker (see Figure 4). We conclude that in zebrafish, Cyp26a1 is expressed at the time and place necessary to provide an RA-degrading function equivalent to Cyp26b1 in tetrapods. These results suggest independent subfunction partitioning of ancestral <i>cyp26</i> regulatory elements in lineages leading to zebrafish and mouse. Arrows point to examples of expressing cells. Dashed lines outline gonads. Scale bar: 0.05mm.</p

Expression of genes encoding enzymes for the synthesis and degradation of RA during zebrafish gonad development.

<p><i>In situ</i> hybridization on adjacent sections of animals representing the three key stages of gonad development: (A–E: n=7) bipotential, sexually undifferentiated gonads with an ovary-like morphology at 20 days post-fertilization (dpf); (F–O) gonads transitioning to testes or ovaries (26 dpf) (F-J: n=4; K–O: n=3); (P–Y and Z–I’) gonads sexually differentiated but still immature (33 dpf and 41 dpf) (P–T: n=3; U–Y: n=4; Z-D’: n=2; E’–I’: n=2). Images show expression patterns of the gene encoding the RA-synthesizing enzyme Aldh1a2 (A, F, K, P, U, Z, E’), the gene encoding the RA-degrading enzyme Cyp26a1 (C, H, M, R, W, B’,G’) and combined probes for <i>cyp26b1</i> and <i>cyp26c1</i> (E, J, O, T, Y, D’, I’) together with the early male gonadal marker <i>amh</i> (<i>anti-Müllerian hormone</i>: B, G, L, Q, V & A’, F’) and the germ-line specific marker <i>vasa</i> (D, I, N, S, X, C’, H’) at four different stages. Expression of <i>aldh1a2</i> was detected in somatic cells in both male and female gonads throughout development (A, F, K, P, U, Z, E’). Expression of <i>aldh1a3</i> was not detected at all in gonads but was detected in retina cells (data not shown) and the ortholog of <i>Aldh1a1</i> was lost in the teleost lineage [54–56]. The expression pattern of <i>cyp26a1</i>, however, showed a distinct sexual dimorphism, as gonadal somatic cells from males (H, R and B’), but not from females (M, W, G’), up-regulated its expression during gonad development. Interestingly, in females, oocytes that had transitioned from early stage IB (yellow arrowhead in M,W,G’) to late stage IB (green arrowheads in M,W,G’) up-regulated the expression of <i>cyp26a1</i> in the ooplasm, which was maintained at later stages (e.g. red arrowhead stage II in G’). The observed expression pattern of <i>cyp26a1</i> in oocytes is compatible with a function in inhibiting meiotic progression and facilitating the meiotic arrest at diplotene stage. Expression of <i>cyp26b1</i> and <i>cyp26c1</i> in gonads was not detected at any of the stages analyzed in either sex (E, J, O, T, Y, D’, I’) Arrows point to examples of expressing cells. Scale bar shown per each row: 0.1mm.</p

Complementary expression of the meiotic recombination marker <i>sycp3</i> and the pluripotent marker <i>pou5f1</i>(oct4) in developing gonads.

<p>In bipotential gonads at 20 dpf, comparison of the expression of the germ cell marker <i>vasa</i> (A), the synaptonemal complex marker <i>sycp3</i> (arrow in B) and the pluripotent gene <i>pou5f1</i> (arrow in C) revealed that <i>sycp3</i> and <i>pou5f1</i> were both expressed in germ cells but in a complementary non-overlapping fashion (red arrowheads and dashed lines in B and C). In differentiating testes at 24 dpf, expression of <i>vasa</i> was detected in germ cells (D) revealing that only some of the germ cells expressed <i>sycp3</i> (arrows in E) but none of them expressed <i>pou5f1</i> (F). In differentiating ovaries at 24 dpf, <i>vasa</i> labeled germ cells (G), and <i>sycp3</i> only labeled those germ cells that were small (arrows in H), which interestingly did not express <i>pou5f1</i> (red arrowheads and dashed lines in I) and had not reached the late stage IB. Complementarily, the larger oocytes that had reached diplotene stage expressed <i>pou5f1</i> (arrow in I). Scale bar indicated per each raw: 0.1mm.</p

Three color fluorescent detection of <i>cyp26a1</i> and <i>vasa</i> expression during zebrafish gonad development.

<p>Fluorescence detection of <i>cyp26a1</i> (green) expression and <i>vasa</i> (red) expression by double <i>in situ</i> hybridization on gonad sections at the bipotential gonad stage at 15 dpf (A: n=1) and 20 dpf (B: n=2), and on immature gonads developing into testes (C: n=1) or ovaries (D: n=1) at 33 dpf. <i>Cyp26a1</i> expression occurs in somatic cells and does not co-localize with the germ cell marker <i>vasa</i> in bipotential gonads (A,B). In immature testes, <i>cyp26a1</i> expression was also expressed in somatic cells (arrow) and not in germ cells (C). In immature ovaries, however, <i>cyp26a1</i> was expressed in large oocytes that had reached diplotene (arrowhead in D).</p

Expression patterns of <i>cyp26</i> gene family members in late developing retina are conserved between zebrafish and mouse.

<p>To learn if the divergent subfunction partitioning between zebrafish <i>cyp26a1</i> and mouse <i>cyp26b1</i> in gonad development applies to other organs, we studied the expression of <i>cyp26</i> paralogs during retina development in 15-dpf zebrafish. Results revealed expression of <i>cyp26a1</i> and <i>cyp26c1</i> in different layers of the retina, but no detectable expression of <i>cyp26b1</i> (A: n=1). This result agrees with the <i>Cyp26</i> expression profile described in mouse retina (B), in which <i>Cyp26a1</i> and <i>Cyp26c1</i> genes were expressed in the inner nuclear layer during post-natal eye development while <i>Cyp26b1</i> was not expressed in the eye [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073951#B74" target="_blank">74</a>]. We conclude that independent subfunction partitioning related to gonad development occurred after the teleost-tetrapod lineage divergence, while subfunctions related to at least one other organ, the retina, are still conserved between <i>Cyp26</i> orthologs in different vertebrate lineages (B).</p

Expression of <i>aldh1a2</i> and <i>cyp26a1</i> is independent of germ cell signaling.

<p>Animals depleted of germ cells that develop into sterile males were generated by <i>dead end</i> (dnd) morpholino knockdown to study the expression of <i>aldh1a2</i> and <i>cyp26a1</i> in gonads at (A-C: n=1) bipotential stage (19dpf), (D–F: n=1) transitioning stage (25dpf), and (G-I: n=1) differentiated testes (37dpf). Results showed that <i>aldh1a2</i> was widely expressed in somatic cells of the gonads at the three different stages analyzed (A,D,G) while <i>cyp26a1</i> expression was detected in a subset of somatic cells also in the three stages analyzed (arrows in B, E, H). These results demonstrate that the onset as well as the maintenance of <i>aldh1a2</i> and <i>cyp26a1</i> expression, at least until 37dpf, is independent of germ cell signaling. Expression of the germ cell specific marker <i>vasa</i> was not detected at any stage (C,F,I), confirming the total depletion of germ cells by <i>dnd</i> morpholino injection in all animals. Gonads are outlined by a dashed line (B,C, E, F,H,I). Scale bar: 0.05 mm (A).</p

Expression patterns of <i>aldh1a2</i>, <i>cyp26a1</i>, <i>cyp26b1</i> and <i>cyp26c1</i> in adult gonads.

<p>In adult testes (n=3), <i>aldh1a2</i> expression was detected in somatic cells surrounding cysts in a localization expected for Sertoli cells (A) and <i>cyp26a1</i> expression was detected in a subset of cells in a localization expected for Leydig cells (B). The presence of germ cells expressing <i>cyp26a1</i> could not be discarded. Expression of <i>cyp26b1</i> (C) and <i>cyp26c1</i> (D) was not detected in adult testes. In adult ovaries (n=2), <i>aldh1a2</i> was detected in somatic cells but not in the oocytes (E,I). Expression of <i>cyp26a1</i> was restricted to the ooplasm of oocytes and was not detected in somatic cells (F,J). In oocytes, <i>cyp26a1</i> expression varied according to the stage of meiosis: it was barely detectable in early stage IB oocytes (<i>e</i>IB, yellow arrowhead in F, prior to the diplotene stage of meiosis), was up-regulated in late stage IB oocytes that entered meiotic arrest at diplotene stage (<i>l</i>IB, green arrowhead in F), was maintained in stage II (red arrowhead in F) and stage III oocytes (green arrowhead in J) and was not detected in stage IV oocytes (J) coinciding with the resumption of meiosis I. Expression of <i>cyp26b1</i> was detected solely in a subset of cells in the somatic tissue surrounding the oocytes (arrows in G, K) and no expression of <i>cyp26c1</i> was detected in the ovary (H,L). Scale bar: 0.1mm.</p