Formation of the genital ridges is preceded by a domain of ectopic Sox9-expressing cells in Lepidochelys olivacea

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Formation of the genital ridges is preceded by a domain of ectopic Sox9-expressing cells in Lepidochelys olivacea
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  Evolution and Developmental Control Formation of the genital ridges is preceded by a domain of ectopic Sox9-expressingcells in  Lepidochelys olivacea Verónica Díaz-Hernández  a , Alejandro Marmolejo-Valencia  b , Martha Harfush  c , Horacio Merchant-Larios  b, ⁎ a Departamento de Embriología, Facultad de Medicina, UNAM. México DF, Mexico City, Mexico b Departamento de Biología Celular y Fisiología, Instituto de Investigaciones Biomédicas, UNAM. México DF, Mexico City, Mexico c Centro Mexicano de la Tortuga CONANP-SEMARNAT, Oaxaca, Mexico City, Mexico a b s t r a c ta r t i c l e i n f o  Article history: Received for publication 2 July 2011Revised 22 September 2011Accepted 1 October 2011Available online 8 October 2011 Keywords: Bipotential gonadSea turtleSox9CytokeratinCoelomic epitheliumGonadal evolution Bipotential gonads represent the structural framework from which alternative molecular sex determinationnetworks have evolved. Maintenance of Sox9 expression in Sertoli cells is required for the structural andfunctional integrity of male gonads in mammals and probably in most amniote vertebrates. However, spatialand temporal patterns of Sox9 expression have diversi fi ed along evolution. Species with temperature sex de-termination are an interesting predictive model since one of two alternative developmental outcomes, eitherovary or testis occurs under controlled laboratory conditions. In the sea turtle  Lepidochelys olivacea , Sox9 isexpressed in the medullary cords of bipotential gonads when incubated at both female- or male-promotingtemperature (FT or MT). Sox9 is then turned off in presumptive ovaries, while it remains turned on in testes.In the current study, Sox9 was used as a marker of the medullary cell lineage to investigate if the medullarycords originate frommesothelialcells atthe genital ridges where Sox9 isupregulated,or, if theyderive from acellpopulation speci fi edatan earlier developmental stage, whichmaintainsSox9 expression. Usingimmuno- fl uorescence and in situ hybridization, embryos were analyzed prior to, during and after gonadal sex deter-mination. A T-shaped domain (T-Dom) formed by cytokeratin (CK), N-cadherin (Ncad) and SOX9-expressing cells was found at the upper part of the hindgut dorsal mesentery. The arms of the T-Dom wereextended to both sides towards the ventromedial mesonephric ridge before the thickening of the genitalridges, indicating that they contained gonadal epithelial cell precursors. Thereafter, expression of Sox9 wasmaintained in medullary cords while it was downregulated at the surface epithelium of bipotential gonadsin both FT and MT. This result contrasts with observations in mammals and birds, in which Sox9 upregulationstarts at a later stage in the inner cells underlying the Sox9-negative surface epithelium, suggesting that theestablishment of a self-regulatory Sox9loop required for Sertoli celldetermination has evolved. TheT-shapeddomain at the upper part of the hindgut dorsal mesentery found in the current study may represent the ear-liest precursor of the genital ridges, previously unnoticed in amniote vertebrates.© 2011 Elsevier Inc. All rights reserved. Introduction Sex determination implies alternative branching in response to dif-ferent initiatingsignals.Onceasignal hasinitiated oneparticularsexualnetwork,adevelopmentalsequenceofgeneactivityispursued.Howev-er, in somatic cells of the morphologically undifferentiated gonads, sexdetermination occurs regardless of the signal that initiates the commit-menttoformeithertestesorovaries.Therefore,theearlygonadal anlage represents the structural framework around which alternative molecu-lar sex determination networks have evolved. Prior to evident sexuallydimorphic histologicalrearrangementsleadingto testisor ovary forma-tion, bipotential gonads of tetrapoda (Amphibia, Reptilia, Aves andMammalia) are formed by two tissue compartments: stromal and epi-thelial. The former comprises blood vessels and connective tissue pre-cursors and, the latter, epithelial-like cells surrounding the incomingprimordial germ cells. The rearrangement pattern of the two compart-ments has evolved, which has led to variability among species in boththe cell autonomous molecular networks and the cell-cell signaling in-teractions underlying the process of gonadal sex differentiation.In mice, the epithelial compartment contains Sertoli and follicularcell precursors in male and female genital ridges, respectively. Trans-gene experiments haveshown that Sox9expressionin preSertolicellsis necessary for testis differentiation; its failure to be expressed leadsto male to female sex reversal (Barrionuevo et al., 2006; Bishop et al.,2000; Vidal et al., 2001). Sox9 expressesin early genitalridges of bothsexes but in genetic females it is soon down regulated (Pask et al.,2010).  Sry  and  Sf1  autonomously activate  Sox9  in preSertoli cellsprior to gonadal differentiation (Sekido and Lovell-Badge, 2008). Since  Sry  is brie fl y expressed while  Sox9  upregulation remains, the Developmental Biology 361 (2012) 156 – 166 ⁎  Corresponding author at: Departamento de Biología Celular y Fisiología, Institutode Investigaciones Biomédicas, Universidad Nacional Autónoma de México, CiudadUniversitaria, CP. 04510, México DF, Mexico City, Mexico. E-mail address:  merchant@servidor.unam.mx (H. Merchant-Larios).0012-1606/$  –  see front matter © 2011 Elsevier Inc. All rights reserved.doi:10.1016/j.ydbio.2011.10.001 Contents lists available at SciVerse ScienceDirect Developmental Biology  journal homepage: www.elsevier.com/developmentalbiology  lattergenefunctions astheeffectorthatsetsuptheSertolicelllineagedetermination program from which both the onset of morphogenesisand the maintenance of functional testis depend (Wilhelm et al.,2007). Recent evidence has shown that the ovarian pathway dependson  Foxl2  and  ER2 , whichmaintain Sox9 silent infollicular cells. If thesemechanisms fail, follicular cells transdifferentiate into Sertoli cells in-ducing postnatal ovaries to form testis-like gonads (Uhlenhaut et al.,2009). Thus, at cellular level, mammalian sex differentiation dependson setting up the male or female network in the supporting epithelialcell lineage.During mouse gonadal morphogenesis, the coelomic epitheliumcontributes to somatic cell precursors in both the XX and the XY gonad during the bipotential stage of development. However, earlymouse gonads do not show cord-like structures at the bipotentialstage (Karl and Capel, 1998).  Sox9  is not expressed in cells of the me-sothelium overlying the undifferentiated gonads, and only begins tobe expressed in Sertoli cell precursors atthe genitalridge core (Bullejosand Koopman, 2004; Moreno-Mendoza et al., 2003). Thus, transcrip-tional activators and/or speci fi c  Sox 9  repressors must be present earlyat the onset of the expression pattern of gonadal cells according withtheir position in the undifferentiated gonad. Considering that cells of the genital ridge are in rapid proliferation, it is of interest to determinethe mechanisms responsible of the spatial regulation of   Sox9  in the di-verse cell lineages within the gonad.Asmentionedabove,inmice,the Sry/Sox9 -expressingcellprecursorsderive from proliferation of mesothelium cells at the genital ridge. Ge-netic females lacking  Sry  brie fl y express  Sox9  but the encoded proteinremains cytoplasmic and is soon downregulated (da Silva et al., 1996). Thus, apparently  Sox9  is expressed in the early genital ridge of bothsexes prior to sex determination. Although in the fast-developingmousegonadsthetwoclassicterritories,cortexandmedulla,canhardlybeidenti fi ed,thesespatialdomainsareformedinthebipotentialgonadsof all tetrapod vertebrates (amphibian, fi shes, birds and mammals).Initiating signals of sex determination have diversi fi ed during ver-tebrate evolution. In therian mammals, in addition to  Sry ,  DmY   and DmW   have also been found; the  fi rst, in two species of teleost  fi sh(Kondo et al., 2003; Matsuda, 2002) and, the second, in the toad  Xenopus laevis  (Yoshimoto, 2008). In birds a dosage effect of   Dmrt1 has been proposed. Testis of   Dmrt1  knockdown male chick embryosdeveloped as ovarian-like gonads in which  Sox9  expression wasstrongly reduced (Smith et al., 2009). In all vertebrates studied sofar, upregulation of   Sox9  plays a key role in testis determinationand/or testis differentiation (da Silva et al., 1996; Jakob and Lovell- Badge, 2011; Merchant-Larios et al., 2010). Maintenance of   Sox9  ex-pression in Sertolicells isrequired to keep thestructuraland functionalintegrity of male gonadsin mammals andprobably in most vertebrates.However,spatialandtemporalpatternsof  Sox9 expressionincellsofthegenital ridges have diversi fi ed during evolution. While mammals up-regulate  Sox9  prior to  Amh  expression (De Santa Barbara et al., 1998)andmorphologicaldifferentiation,theoppositeoccursinbirdsandcroc-odiles,inwhich Sox9 isup-regulatedafter  Amh expressionandmorpho-logical differentiation of testis cords (Oreal et al., 1998; Western et al.,1999a, 1999b). Species with temperature sex determination (TSD) are interestingpredictive models since one of two alternative developmental out-comes occur under controlled laboratory conditions. Thus, consecutivemolecular patterns of gene expression underlying morphogenesis maybe studied before, during and after morphological changes become ev-ident. Previous results in the sea turtle  Lepidochelys olivacea  showedthat  Sox9  expresses in the somatic cells of medullary cords of undiffer-entiated gonads incubated at both female-promoting temperature(FPT) and male-promoting temperature (MPT) (Moreno-Mendoza etal., 1999). Thereafter,  Sox9  is turned off prior to splicing of medullarycords in presumptive ovaries of embryos incubated at FPT, while Sox9continuestobeexpressedintestesatMPT.AsimilarpatternofSox9ex-pression was found in two other species with TSD:  Trachemys scripta (Barske and Capel, 2010) and  Alligator mississipiensis  (Western et al.,1999b). Therefore, since Sox9 expression is independent of tempera-tureinearlygenitalridges,itcanbeusedasamarkerofthecelllineage,which forms the medullary cords prior to sex determination.The aim of this study was,  fi rstly, to detect the  Sox9  expression pat-tern in gonads of   L. olivacea byimmuno fl uorescence and  in situ  hybrid-ization,prior, duringand after gonadal temperature sexdetermination,thereby directly addressing the question of whether the medullarycords srcinate from mesothelial cells that up-regulate Sox9 or if theyderive from an early speci fi ed cell lineage that maintains Sox9 expres-sion. Secondly, we sought to assess if Sox9-expressing cells maintaintheirepithelialidentityfromtheonsetofgenitalridgedevelopmenton-wards in this species. Materials and methods  Animals and sample collection This study was approved by the local Ethics Committee at the Insti-tutodeInvestigacionesBiomédicas,UNAM.Eggsof  L.olivacea werecol-lectedatLaEscobillaBeach,Oaxaca,Mexico,and transportedtoMexicoCity within 12 h after collecting. The eggs were kept in boxes withmoistened vermiculite and incubated either at 26±0.5 °C or at 33±0.1 °C, male- (MPT) or female-promoting temperature (FPT), respec-tively. The experiments were performed using eggs from four clutchesand the developmental stages were determined according to Miller'scriteria (Miller, 1985). Embryos were collected prior to the establish-mentoftheurogenitalridgeatstage20,atstages21 – 25,theundifferen-tiated gonads period, and at stage 27, when ovaries and testes areevident. BrdU administration Eggswereilluminatedtolocatethebloodvesselringaroundtheem-bryo and injected with 100 g/g of egg of 5-bromo-2´-deoxy-uridine(Sigma Aldrich), and the embryos removed 1 h after the injection. Immuno  fl uorescence An antigen-af  fi nity antibody raised against the SOX9 C-terminal24-aminoacidepitope(VPSIPQTHSPQHWEQPVYTQLTRP)thatprovedto be highly speci fi c against the SOX9 of   L. olivacea  was prepared aspreviously described (Moreno-Mendoza et al., 2001) Fixed embryoswere washed in PBS and dehydrated with 30% sucrose overnight,1:1 30% sucrose: O.C.T compound (Tissue Tek). The specimens wereembedded in O.C.T compound and frozen at  − 20 °C. SOX9 proteinwasdetectedin10 – 25  μ  mfrozensections.Toremovelipids,theslideswere washed with ascending and descending series of ethanol in PBS.Antigens were retrieved by boiling the slides in citrate buffer, pH 6.0,for 45 min and allowing slides to cool to room temperature (RT).Slides were washed with PBS and then permeabilized with 0.5% Tri-ton 100X /PBS for 10 min. To block unspeci fi c staining the sectionswere treated with 5% horse serum in PBT (0.5% Triton 100X/PBS) for2 h at RT and subsequently incubated overnight with primary rabbitanti-Sox9 antibody diluted 1: 150 at 4 °C. The sections were incubatedin secondary goat Cy3 anti-rabbit antibody (Chemicon International,Inc.) diluted 1:100 for 1 h RT and washed with PBS, sequentiallyblocked with 1% horse serum in PBS for 1 h, and incubated overnightwith mouse anti-pan cytokeratin (AE1/AE3+8/18) (Biocare Medical)whichreactswithacommonepitopetoacidandbasiccytokeratins(di-luted 1:100 at 4 °C) or with mouse anti-N-cadherin (Invitrogen). Theslides were washed and incubated in Alexa  fl uor 488 donkey anti-mouse secondary antibody (Molecular Probes, Invitrogen) (diluted1:100) for 1 h RT.For Sox9/BrdU double immuno fl uorescence, the lipids were re-moved from the slides with alternate serial sections as described 157 V. Díaz-Hernández et al. / Developmental Biology 361 (2012) 156 – 166  above. The sections were immersed in 2 N HCl at 37 °C for 30 min todenature DNA and incubated with 10 mM borate buffer pH 8.5 at RTfor 15 min to neutralize HCl. To block unspeci fi c staining, sectionswere treated with 5% horse serum in PBT (0.5% Triton 100X/PBS) for2 h at RT and subsequently incubated overnight with primary rabbitanti-Sox9 antibody diluted 1: 150 at 4.0 °C, sequentially blockedwith 5% horse serum in PBS for 1 h, and incubated overnight withmouse anti-BrdU diluted 1:250 (Roche). Slides were washed and in-cubated in Alexa  fl uor 488 donkey anti-mouse secondary antibody(Molecular Probes, Invitrogen) (diluted 1:100) for 1 h RT. All sectionswere counterstained with TOTO-3 iodide (Molecular Probes, invitro-gen). To test the immunostaining speci fi city of the SOX9 antibody, theantibody and the antibody plus the blocking peptide were analyzedbyWesternblot(seesupplementarymaterialandmethodsandSupple-mental Fig. 2). As negative controls in the frozen sections, the antibodywasomittedintheprimarysolution,orpeptideimmunogenwasaddedtothesolutioncontainingtheSOX9antibody(SupplementalFig.1).Im-ages were collected and processed with a confocal Zeiss Pascal LSM5microscope. Light and electron microscopy The samples were processed for high-resolution light and electronmicroscopyas previously describedin(Merchant, 1975).Brie fl y, sam-ples were  fi xed in Karnovsky buffer, post fi xed in 1% OsO 4 in Zetterq-vist's buffer, and embedded in Epon 812. Semithin (1 m) and thin(60 nm) sections were stained with toluidine blue and uranyl ace-tate/lead citrate, respectively. Images were collected and processedwith a light or transmission electron microscope. Whole mount in situ hybridization Turtle SOX9 fragments of 597 bp were generated by PCR ampli fi -cation of cDNA from MPT gonads at stage 27. PCR primers were:SOX9 F: 5 ′  AGG AAG TCGGTG AAG AAC G 3 ′ ; SOX9-R: 5 ′  CTT GATGTG TGT CCTCTG CTG 3 ′ . The PCR product was cloned into thepGEM-T easy vector system I (Promega corporation). Fragment iden-tity and insertion orientation was veri fi ed by sequencing. Plasmidscontaining the turtle SOX9 fragmentwere linearized by restrictiondi-gestion and used as templates for either SP6 or T7 RNA polymerase(Promega).  In vitro  reactions generated antisense and sense ribop-robes, and these were labeled with digoxigenin-UTP (DIG RNA label-ing mix, Boehringer).  In situ  hybridization was performed as reported(Díaz-Hernández et al., 2008) Test or control samples were hybrid-ized with antisense or sense riboprobes, respectively. Riboprobeswere added and hybridized overnight at 56 °C, washed and blockedwith 15% goat serum, 2% BSA in PBS. The signal was immunologicallydetected using preabsorbed antibody anti-digoxigenin-AP Fab frag-ment (Boehringer Mannheim), and staining with BM Purple AP Sub-strate precipitating (Roche). Samples were photographed using adissecting microscope. Results The cortex and medullary cords are two epithelial gonadal compartments Keratins are intermediate cytoskeleton fi laments typical of epithe-lial cells. In the current study, a pancytokeratin antibody that detectsseveral members of the keratin family was used to identify the epithe-lial lineage of developing genital ridges. At stage 28, more than 90% of the gonads from embryos incubated at either male- (MPT=26 °C) orfemale-promotingtemperature(FPT=33 °C)showedmorphologicallydifferentiated testes or ovaries, respectively. Double immunostainingrevealed testis with medullary cords formed by cells with SOX9-expressing nuclei and containing cytokeratins in the cytoplasm.The surfaceepithelium ofmalegonadswasformed bya single layerof squamous cytokeratin-positive (CK + ) cells lacking SOX9 expression(SOX9 − ) (Figs. 1A – C). On the other hand, epithelial tissues in ovariesshowed a dramatic rearrangement compared to testes. The surface ep-ithelium was thicker and formed by several layers of tightly attachedCK + columnarcells,while the medullary cords appeared assingle clus-ters of CK − epithelial cells distributed in the ovarian medulla (Figs. 1D,E). These epithelial clusters derive from the splitting up the medullarycords of previously undifferentiated gonads in which SOX9 was gradu-ally turned off at FPT (Fig. 2A).Up to stage 24, gonads from embryos incubated either at MPT orFPT remained morphologically similar: one or two layers of SOX9 − cubic cells forming the surface epithelium and the medulla occupiedby SOX9 + cells forming a network of medullary cords. Both types of CK cells appeared continuous at several locations (Figs. 2A – B). Thus,differential SOX9expressioninthe cortexand medulla allowsdistinc-tion between two kinds of epithelial cells in the morphologicallyundifferentiated genital ridges of   L. olivacea .  Autonomous proliferation of SOX9 + cells of medullary cords and SOX9 − cells of the surface epithelium If SOX9 − cellsof thesurface epitheliumcontain precursorsofSOX9 + cells, then, asymmetric cell division of these precursor cells may explainthe srcin of the medullary cords. Since nuclear labeling with BrdU canmark differential proliferative rates in developing organs with twowell-distinguishedterritories,thisDNAprecursorwasinjectedtoembry-os at stages 22 – 23 whose gonads were still morphologically undifferen-tiated. At stage 22, a cranio-caudal gradient of SOX9 ÷ BrdU + cells wasfound at the surface epithelium (mesothelium). Along the caudal thirdof the genital ridges most cells incorporating BrdU were SOX9 − (Fig. 3Aand Supplemental video 1); their number decreased at the medial third(Fig. 3B), while at the cranial third most BrdU + were SOX9 − . Once thecomplete genital ridge was established at stage 23, gonads were foundto grow by autonomous proliferation of SOX9 + and SOX9 − epithelialcells at the medulla and cortex, respectively (Supplemental Fig. 3A).To  fi nd out when regional allocation of SOX9 + -CK + and SOX9 − – CK + cells appears, we studied earlier genital ridges. Thus, stages 20and 21, which are prior and at the onset of the establishment of gen-ital ridges, respectively, were processed. At early stage 20, formationof the dorsal mesentery occurs by a process of in-folding at bothsides of the mesothelium separating the left and the right sides of the embryo (Figs. 4A and E). Cytokeratin staining revealed CK + epi-thelial cells at several locations: in the mesothelium bordering thecoelomic cavity, the mesonephric ducts, condensing mesonephric tu-bules, the notochord, and the endoderm of the vitelline sac (Fig. 4B).SOX9 plays a critical role as transcription factor for speci fi cation andmaintenance of several organs in metazoan (Wegner, 1999). Thus,as described in other tetratopoda (Guth and Wegner, 2008), SOX9-expressing cells appeared distributed in several developing organsof   L. olivacea , including the neural tube, notochord and mesonephros.Interestingly, a new patternof SOX9 expression was found, whichhasnot been previously described, CK + mesothelial cells along the lateralfolds of the just forming dorsal mesentery, strongly expressed SOX9priortoformationof genitalridges(Fig.4CandSupplementalFig.3B).  A population of SOX9 + epithelial cells at the dorsal mesentery precedesthe establishment of the genital ridges Asthedorsalmesenterygrew,atlaterstage20andearly21,apatternofSOX9 + cellsappearedamongtheCK + tissues.CellswithSOX9 + nucleiwere distributed in the upper part of the dorsal mesentery and in themesothelium that covers the mesonephric ridges along the area inwhich the genital ridges will later be formed (Figs. 5B and E). SOX9 + cells were not restricted to the surface epithelium but were also ob-served in the inner upper part of the dorsal mesentery as less tightly 158  V. Díaz-Hernández et al. / Developmental Biology 361 (2012) 156 – 166  arranged CK + cells resembling mesenchymal cells (Fig. 5E). Together,theSOX9 + cellsforma “ T ” shapeddomain(T-Dom)asseenincrosssec-tions of the posterior part or the embryo. The arms of the T-Dom delin-eate the mesonephric mesothelium where the genital ridges will laterbe organized (Fig. 5).Due to the growth of the adjacent mesonephros, stage 21 embryoserial sections showed that the T-Dom SOX9 + cells were now locatedbetween the two urogenital ridges. At this stage, the arms of the T-Dom were folded down at each side of the hindgut mesentery andthe CK + – SOX9 + cells occupied a thicker layer on the surface epithe-lium (Figs. 6A – D). Serial sections of the anterior part of the T-Domshoweda prominence of the thicker epithelium into the coelomic cavi-ty, whichmarked the onsetof the genital ridge formation(Supplemen-tal Fig. 4 and Supplemental video 2). Most mesenchymal-like cellsunderlying the surface epithelium at both, the upper part of the dorsalmesentery and the incipient genital ridges, were CK + – SOX9 + suggest-ing their epithelial srcin (Figs. 6B – D).Although CK + – SOX9 + cells showed a spatial pattern suggesting aprecocious speci fi cation of the epithelial component of genital ridges(surface epithelium and medullary cords), both cell markers wereexpressed in cell lineage precursors of other organs that share a com-mon mesodermal srcin with the genital ridges (namely, the meso-nephric tubules and adrenal cell cortex). Next, another cell markerwas investigated to reinforce the idea of a putative pregonadal do-mainofgenitalridgeepithelialcellprecursors. Twoantibodiesagainstcell adhesion molecules (CADs) were tested: E-cadherin (Ecad) andN-cadherin (Ncad). Ecad was slightly detected in PGCs and in allCK + somatic cells (not shown) but strong Ncad staining colocalizedwith the CK + – SOX9 + cells of the T-Dom at stages 21 and 22(Fig. 7A, E; Figs. 8A – E and Supplemental Fig. 5 AB). Thus, this strongN-cadherin expression delimits the spatial pattern of the putativeCK + – SOX9 + cell population lineage, which may be involved in theestablishment of the epithelial tissues that form the genital ridges.To correlate the expression of SOX9 protein shown by our anti-body with  Sox9  transcripts, an  in situ  hybridization (ISH) was per-formed. Although  L. olivacea  embryos were quite resistant to probepenetration in whole mounted samples,  Sox9 -positive tissues wererevealed on the surface of thick-sectioned embryos prior to ISH. Dis-tribution of   Sox9 -positive tissues closely matched the SOX9 + proteincells detected by immuno fl uorescence (compare Figs. 6 and 9). Thedorsal mesentery and ventromedial surface of the mesonephrosshowed a T-Dom formed by  Sox9 -stained cells similar to the patternseen with immuno fl uorescence positive tissue at its upper part, andunstained at the lower part towards the vitelline sac and the hindgut(Figs. 9C and D). As the coelomic epithelium thickens at the ventro-medial mesonephros on each side of the dorsal mesentery,  Sox9  tran-scripts were clearly detected at the genital ridges (Fig. 9D).To study details of cell arrangement in the T-Dom, samples wereprocessed for high-resolution microscopy. Semithin sections (1  μ  m) of plastic-embedded tissues showed that, in contrast with the  fl at cells of the coelomic epithelium surrounding the mesonephros, epithelial cellsat the T-Dom appeared tightly arranged, with cubic or columnar Fig. 1.  Differentiated gonads of   L. olivacea  at stage 28. A. Testis showing medullary cords (mc) formed by CK + cells (green) with SOX9 + nuclei (purple). Stromal cells (st) and sur-face epithelial cells (se) show blue nuclei stained with TOTO-3 iodide. Notice the heavier deposit of cytokeratin in the medullary cords at the mesonephric side (*). B. SOX9 and C.cytokeratin D. Ampli fi cation of the ovary. The surface epithelium is formed by more than one layer of tightly attached CK + columnar cells (se). Arrowheads point to fragmentedmedullary cords formed by clusters of CK + cells (arrowheads) randomly distributed among CK − stromal cells. E. Show a general view of the ovary. Scale Bar A – D: 20  μ  m, E=50  μ  m.159 V. Díaz-Hernández et al. / Developmental Biology 361 (2012) 156 – 166  Fig. 2.  SOX9 and CK in genital ridges of sea turtle at stage 24 incubated at female-promoting temperature (FT) or male-promoting temperature (MT.) A. Cross section of a genitalridge from an embryo incubated at MT. Medullary cords (mc) formed by SOX9 + cells (red) and the surface epithelium (se) formed by SOX9 − cells (blue) are shown. Cytokeratinappears as irregular green spots distributed both at the surface epithelium and medullary cords. B. Higher magni fi cation of the same sample shown in A. The antibody against SOX9stains nuclei of medullary cells (red+blue=purple) except for the nucleoli which appear as dark spots (arrow heads). Notice that CK (green) is seen as layers or irregular spotsaround SOX9 + and SOX9 − nuclei of epithelial cells. C. At FT most nuclei of medullary cords are still SOX9 + , similar to gonads from embryos at MT. At the mesonephric side a clusterof purple cells inside a blood vessel (bv) is shown at the upper right corner. Since these cells are similarly seen in pre-adsorbed controls (see Supplemental Fig. 1), they cannot beconsidered SOX9 + cells. D. Higher magni fi cation of the same sample as shown in panel C. Distribution of cells with SOX9 + and SOX9 − nuclei in medullary cords (mc) and surfaceepithelium (se), respectively, is similar at both temperatures. Cytokeratin arrangement indicates continuity between epithelial cells at the surface and medullary cells. Scale bars: Aand C=50  μ  m. B and D=10  μ  m.SOX9 and CK in genital ridges of sea turtle at stage 24 incubated at female-promoting temperature (FT) or male-promoting temperature (MT.) A.Cross section of a genital ridge from an embryo incubated at MT. Medullary cords (mc) formed by SOX9 + cells (red) and the surface epithelium (se) formed by SOX9 − cells (blue)are shown. Cytokeratin appears as irregular green spots distributed both at the surface epithelium and medullary cords. B. Higher magni fi cation of the same sample shown in A. Theantibody against SOX9 stains nuclei of medullary cells (red+blue=purple) except for the nucleoli which appear as dark spots (arrow heads). Notice that CK (green) is seen aslayers or irregular spots around SOX9 + and SOX9 − nuclei of epithelial cells. C. At FT most nuclei of medullary cords are still SOX9 + , similar to gonads from embryos at MT. Atthemesonephricsideaclusterofpurplecellsinsideabloodvessel(bv)isshownattheupperrightcorner.Sincethesecellsaresimilarlyseeninpre-adsorbedcontrols(seeSupplementalFig. 1), they cannot be considered SOX9 + cells. D. Higher magni fi cation of the same sample as shown in panel C. Distribution of cells with SOX9 + and SOX9 − nuclei in medullary cords(mc) and surface epithelium (se), respectively, issimilar at both temperatures. Cytokeratin arrangement indicates continuity between epithelial cells at the surface and medullary cells.Scale bars: A and C=50  μ  m. B and D=10  μ  m. Fig. 3.  Cross sections along the genital ridges and the hindgut mesentery at stage 23 incubated at MT showing proliferative cells as assessed by BrdU incorporation. A. Immunode-tection of SOX9 in a cross section seen with Nomarsky optics added to the confocal image. Several SOX9 + cells that took BrdU appear in the surface epithelium (arrow heads) at theposterior part of the genital ridges. The hindgut mesentery (hgm) located between the genital ridges is shown. (See supplemental video 1). B. At the anterior part of the genitalridge, most cells labeled with BrdU in the surface epithelium are SOX9 − (arrow heads). Scale bars: A=20  μ  m. B=50  μ  m.Cross sections along the genital ridges and the hindgutmesentery at stage 23 incubated at MT showing proliferative cells as assessed by BrdU incorporation. A. Immunodetection of SOX9 in a cross section seen with Nomarsky opticsadded to the confocal image. Several SOX9 + cells that took BrdU appear in the surface epithelium (arrow heads) at the posterior part of the genital ridges. The hindgut mesentery(hgm) located between the genitalridges isshown.(See supplementalvideo1). B.At the anteriorpart of the genitalridge, most cells labeled with BrdU in thesurface epithelium areSOX9 − (arrow heads). Scale bars: A=20  μ  m. B=50  μ  m.160  V. Díaz-Hernández et al. / Developmental Biology 361 (2012) 156 – 166
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