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An mRNA differential display strategy for cloning genesexpressed during mouse gonad development

KATAR INA NORDQVIS T'.2' and V IR PI T OHONEN '1Department of Cell and Mo lecu la r B io logy, Medical Nobel Institu te, Karolinska Institu ter. Stockholm , Sweden, 2Labora to ry o f

Developmental Genetics, Medical Research Council, National Institu te fo r Med ical Research, London, United Kingdom

ABSTRACT The mRNA d iffe re ntia l d is pla y te ch niq ue h as become a p op ula r m eth od fo r iso latin gnovel genes in a variety of biological systems including carcinogenesis, hormone regulation, plantbiology and neurobiology. W e have further developed the m ethod by optim izing different steps fortheuse of small amounts of material, such that differential display can be used in the study ofdevelopmental biology. Our techniques include a new assay for elimination of false positive cDNAc lo ne s a nd a s em i- qu an tita tiv e r ev ers e tr an sc rip tio n-p olyme ra se c ha in re ac tio n (RT-PCR) m ethod forthe rapid analysis of differences in gene expression. T his im proved mRNA differential display strategyreq uires less th an 4 ~ lg o f to tal RNA. W e have used it for the isolation of genes w hich are expressedduring gonad developm ent in the m ouse. O ne of the cDNAs found, cDNA 4 .3 which corresponds toa part of the gene encoding the steroid hydroxylase 3~HSD I, was shown to be a valuable marker foradrenal developm ent and for Leydig cell differentiation and organization during testis developm ent.KEY WORDS : d if fn nl li n! d i.\ !) !n y, (p jti s d l'1 .J el ojmw lIl , .\f'.\"d l' lt Tl ll ilwt io ll , L I') 'd ig n 'l f d l! fi '1 l' lI li nt io ll , I IIOI l, \I '

IntroductionThe mRNA differential disp lay technique was developed by

Liang and Pardee in 1992 and has been used to isolate novelgenes and to compare gene expression in different tissues ordiffere ntly treated cell lines (re view ed by Lian g and P arde e, 1995 ;McClel land et a/ " 1995). Several hundred articles have beenpublished o n the subject. m ost of them w ith a focus o n tum origen-esis. However, only a few of them describe the application of themRN A differential display technique in deve lopm ental bio logy (fo rexample Zimmermann and Schultz. 1994; Adati e t a l" 1995;Conwa y, 1 99 5; G uim ara es e f a l. , 1 99 5; Dav is e f a l. , 1 99 6; M as onef al " 1996). This can partly be explained by the lim ited am ount ofmRNA which is available from biological material relevant ford evelop mental biology studies a nd by th e relative lack o f tech nicalprocedures which describe the use of m RNA differential disp lay ind ev elo pmen ta l b io lo gy (Z immerman n a nd S ch ultz , 1 99 4; Ik on omovand Jacob, 1996). W e present here an m RNA differential displaym ethod w hich can iso late genes expressed d uring gona d de velop-m ent in m ouse. The m ethod w e develop is generally applicable fors tu dy in g a ny d ev elo pmen ta l p ro ce ss .

T he mRNA d iffe re ntia l d is pla y te ch niq ue p erm its s im ulta ne ou sidentification of both up. and down-regulated genes and is basedo n t he c ompar ativ e a na ly sis of sub populations of cD NA s w hich a reproduced by reverse transcription (RT) and polym erase chainreaction (PCR). I n b rie f. t he mRNA popu la tions f rom two d if fe renttissues or cell lines are divided into four subp opula tions using four+A dd ress for rep rints: D ep artm ent o f C ell an d M olecular B io lo gy , M ed ical N obel In stitute, K aro lin ska In stitu tet, 5 -1 71 77 S tock ho lm , S weden . F A X : 46-8-313529.e-mail : Katarina .Nordqvis [email protected] i .se02 f4-62X2/97/$05.00o e B C P re HP ri nt ed i n < ;r ~i n

different 3 '.anchored oligo(dT) prim ers in four separate R T reac-tions. The cDNA fragm ents are then am plified using the sam e seto f 3 '-a nc ho re d p rim ers in c ombin atio n w ith s ho rt a rb itra rily c ho se n5 '-p rim ers in th e p re se nc e of [Cl.33P]dATP. B etw een 50 and 100cDNA bands, in the size range 100 to 500 bp, are produced in eachdifferential disp lay reaction, representin g the 3' end of the mRN As.The PCR products from the two different tissues or cell lines thathave been am plified with the sam e prim er m ix are then separatedon denaturing polyacrylam ide gels and cDNA bands with differentsign al intensity are can didates fo r clonin g a nd fu rther analysis.

The adult gonads, ovary and testis, emerge from precursortissue s initially common to both fem ales and m ales: the ind ifferentgonad. This anlagen appears as a narrow band of tissue along themesonephros , wh ich is located on the dorsal m esentery, on eitherside o f th e do rsal aorta and neural tube in the em bryo (rev iew ed byMcElreavey ef al.. 1995; Nordqvist. 1995; Ramkissoon andGoo dfe llow, 1 99 6). T he d ev elo pmen t o f th e in diffe re nt g on ad intoeither a testis or an ovary depends on the action of the sexchrom osom es. In 1 990, analysis of a V-chrom osom e specific 35 kbDNA fragment in XX males together with mapping of the sex-,\hIl//1'illliOIl\ I II I' d i ll I hi l Iml"": dpc. da~~ pm! coilum: RT, n '\( 'I ~t' I ra n~ ni l) -lion: PC R, p()]~ II1t'ra~ l' chain H 'M liI1l1:'II), SI'x-dt'lt'rllJining- H 'g-i011 '1"("1110-m n~ on1t" ~t'nt'; \1"1-/, \\'i!m < llJlJlO r )..:"t'111'1: SI~ l. SIl'roidog-t'nic bnor 1: ,"iIlX.S ,)'-rt'l.lIl'd 1 1\IC h m..-c on ta il1 in g- g -e1 1t': Ih,\"-I, D S."i-.\1 H : c ritic,1 1 H 'g -io l1 X .gt:ne 1; 3fJIlSU I, :\~ -h"lr()x~~It'rojd ddl\ drog-l'11a,,' ~ t'IH ' I: CTI' J;, ~Il'roid1 1( 1- h~ d ro xy la ~e g el lt' : IlI'ln; h ~ p ( )x an th il 1l ' p IH )~ pI 1' H il !! ,, ~ I lr al l~ rl 'l" a~ t' ~ en t'.


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F ig. 1. Schem atic flow chart of the m RNA differential display strategy.A detailed description of each step is presented in the text (R esults andE xp erim en ta l P ro to co ls) L ess th an 4 t1g total R NA from control and testtissue is needed for an mRNA differential display screening with fourd iffe re nt 3 '-a nc ho re d o llg o(d T) p rim ers a nd tw en ty d iffe ren t a rb itra ry 5' -p rim ers. T his a lso in clu de s R NA fo r se mi-q ua ntita tiv e R T-P CR a ssa yin g fo rdifferences in t ra ns cr ip ti on le ve ls o f g en es Is ol ate d.

determ ining region of the m ouse chrom osom e led to the isolationof SRY/Sry, S ex-d eterm ining R eg io n Y gen e (G ubb ay e t a l. , 1 99 0;Sinclair e t a l. , 1990 ). This gene is conserved on the Y chro moso meam ong alm ost all m ammals tested. In cases of sex reversal in bothhum ans and m ice, the presence or ab sence of this gene correlatedexactly w ith m ale or fem ale phenotype (review ed by Goodfellowand L ovell-B adge, 1993; K oopm an, 19 95). F urther, X X Iransgenicm ice carrying a 14 kb D NA fragm ent encoding m ouse Sryshowednormal male internal and external genitalia and norm al m atingb eh av io r ( Ko opman e l a l. , 1 99 1) . T hu s, SRY/Sry is the only geneon the Y chromosome which is required for testis formation inmammals.

The fruitfu l com bination of hum an ge netics and m ou se develo p-mental biology has led to the isolation of several other genesim portant for gonad development (reviewed by Schafer, 1995).Two of these other genes, W t-! an d Sf-I, are important for thedevelopm ent of the indifferent gonads in both fem ales and m ales,a s has be en show n by gene ta rgetin g disruptio n (K reid berg e t a t. ,1993; Luo et al., 1994). The expression pattern of Sox-g, incombination with information from hum an m utations w ith a sexreversed phenotype, suggests that this gen e is involve d in S ertolicell d ifferentiation and probably is one member of the group ofg enes w hich orchestra tes testis deve lopm ent (Fo ster et a l. , 1994 ;Wagner et al., 1994; Da Silva et al., 1996; K ent et a l. . 1 99 6).Dax-1 may be im portant for ovary develo pm en t, since duplicationof the chrom osom al region containing this gene appears to over-ride SRY and gives rise to XY females (Bardoni et al., 1994;Zanaria e t a l.. 1 994). In addition, m ouse Dax-I e xp re ss io n c oin -cides w ith a ro le in ovary developm ent (Bae e t a t. , 1 99 6; Ik ed a etal.. 1 99 6; Swain e t a l., 1 99 6).

A ll the genes d escribed above encod e potential transcriptionfactors. It is therefore likely th at transcriptio n o f o ther g enes w hichare im portant for testis or ovary developm ent w ill be affected, andthat their differential expression w ill be detected by the mRNAd iffe re ntia l d is pla y meth od . In o rd er to fin d g en es in vo lv ed in g on addevelopment and sex differentiation we have created an mRNAd ifferential display strategy w hich m akes it possib le to isolate andclone genes from a sm all am ount of tissue (Fig. 1). This strategyinclu des a n optim iza tion of th e curre nt m RN A differentia l displaym ethod, a novel m ethod for avoiding false positive clones and arapid sem i-quantitative R T-P CR m ethod to m ea sure difference s ingene expression. Using our m ethod, less than 4 ~g of tota l RNA isrequired fo r the scree ning f or n ov el g en es w ith 20 d if fe re nt a rb itr ar y5 '- pr ime rs . We hav e cloned cDN A 4.3 and cDN A 80.8from the 13.5dpc testis mRNA pool using our method. These two cDNAscorrespond to two genes which encode steroid hydroxylases,3fJHSD I an d GYP17. cDNA 4.3 was used as a probe in whole-mount in s it u h ybridizations and w as show n to be a m arker foradrenal developm ent and for fetal Leyd ig cell organiza tion duringtest is deve lopment .Experimen ta l P ro tocol sTissue and RNA preparations

Gonads w ith attached m esonephros w ere dissected from em .bryos aged betw een 11.5 and 17.5 dpc, frozen directly onto dry iceand s to re d a t - 70 :C . Geno typic sex was dete rm ined by s ta in ing f ors ex c hroma tin in amn io tic c ell n uc le i a s d es crib ed b y Palme r a ndBurgoyne (1991b) ,or by morphologica lexaminat ion 01the gonads,lo ok ing fo r t e st is c or ds a t 13. 5 dpc and t he re af te r. T o ta l R N A w a s


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i\JOlfse gonad de\'elopm ent and m RNA differential display 62 9

isolated from tissues by the m ethod described by C hom czynskian d S acchi (19 87) w ith some m in or m o di fic at io ns . F or e ac h ti mepoint and sex, at least ten gonads with attached m esonephroswere pooled and 500 pi of solution 0 (25 g guanidinium thiocy-anate, 29.3 m l H20, 1.76 m l 0.75 M Na citrate pH 7.0, 2.64 m 110%sarkosyl, 38 pi 0-m ercaptoethanol) was added. The tissues w erehomogenized by squeezing five times through a 0.8x50 sterileneedle fo llowed b y te n time s th ro ugh a 0.6x25 s te rile n ee dle . T hefollowing solutions w ere added: 50 pi N a acetate (2 M , pH 4.0), 500p i H 20-buffered phenol, 100 pi chloroform /isoam yl alco hol (24:1).T h e m ix t u r e w a s v o r t e x e d fo r 1 m in , c h i l le d o n ic e f o r 1 5 m in a n dc e n t r i f u g e d at 4'C, 12000 rpm, for 15 m in. The upper phase w a sr e m ov e d a n d e x t r a c t e d o n c e w i t h 5 0 0 ~ j c h lo r o f o rm /i s o a m yl a lc o -h o l ( 2 4 :1 ) . T h e R N A w a s p r e c ip i t a te d by addit io n o f 1 m l 9 5 %e th a n o l , t h e p e l le t w a s w as h e d w it h 7 0 % e th a n o l , r e s u s p e n d e d inH 20 and kept at -70'C. Since the RN A concentration and quality iscritical for the fo llo win g mRN A differential disp lay, R NA sam pleswere quantitated by absorbance at 260 nm and analyzed by 1%agarose gel e lectrophores is .D N as e t re atme nt

One pg of total RNA was added to a m ixture of 5 pi DR-buffer(400 mM Tris pH 8.0, 100 mM NaCI, 60 mM MgCI2, 50 mM DTT),1 111RNAsin (40 U/pl, promega, cat.# N2511), 1 pi ROI RNase-free DNase (1 U/pl, Prom ega, cat.# M 6101) in a total volum e of 50pi, incubated at 37'C for 15 m in, extracted once by phenol andprecipitated w ith 10 pg glycogen (Boehringer M annheim , caU 901393). DNase treated RNA was resuspended in H20 and stored at-70'C.Pol y(A ) se le cti on

200 I.g of tota l RNA from female and male 13.5 dpcg on ad s+me so ne ph ro s w ere p oly (A )' s ele cte d b y f he P oly (A ) q uik m RN A p urification kit (S tra tage ne, cat.# 2 0 0 3 4 8 ) . Poly(A)' R NAwas resuspended in 50 pi H20 and 2 pi was used for RT,corresponding to approxim ately 8 pg of total R N A .P r i m e r s

Oligonucleotide prim ers T"CA, T"GC, T"CT, T"AG (denoted3' prim ers) and the arbitrary decam ers (denoted 5' prim ers), 5'2(GCAAGTACCG), 5'3 (CCATGTCACC) and 5'20 (AAGGCCTT-TA ), w ere synthesized by the core facility at N ational Institute fo rM e d ic a l R e s e a r c h , L o n d o n . T h e a r b i t r a r y s e q u e n c e s w e r e se -le cte d ra nd om ly . P rim ers fo r S ry, H PR Tand 3 fJH SD Iwe re syn the-sized by the core facility at CMB, Karolinska Institu te, and ares h o w n b e lo w in 5 ' t o 3 ' d ir e c t io n .

5 'H P R T -C C T G C T G G A T T A C A T T A A A G C A C T G ,3 'H P R T -G T C A A G G G C A T A T C C A A C A A C A A A C ,5 'S r y -G G T T G C A A T C A T A A T T C T T C C ,3 'S r y - C A C T C C T C T G T G A C A C T T T A G ,5 '3 0 H S D I -G A A G C C T T G C C A G T C A C T A A C ,3 '3 0 H S D I-G G C A A G A T A T G A T T T A G G A C .

mRNA d if fe re nt ia l d is pla yRev er se tr an sc rip tion

Fo r each m R N A popu la tion, r ever se fr ansc rip tion was done inf o u r in d e p e n d e n t r e a c t i o n s u s in g th e f o u r d i f f e r e n t 3 ' p r im er s( T 1 1 N N ) . R e a c t i o n s w e r e m ix e d o n ic e , e a c h r e a c t io n c o n t a in in g 2

pg total RNA or 2 pi poly(A)' RNA and 1 pI3'-primer (10 pM) in atotal volume of 14 pI. The RNA was denatured at70'C for 8 m in andt r a n s f e r r e d b a c k to i c e . T h e fo l l o w in g c o m p o n e n t s w e r e t h e nadded to each reaction: 5 pl5xRT buffer (200 m M Tris pH 7.5, 150m M KCI, 15 mM MgCI2), 2.5 pi DTT (0.1 M), 2.5 pi dNTPs (375 pM ),0.5 111rRNasin (40 U/pl, Promega, caU N2511) and 1.0 piSuperScript II RT (200 U/pl GIBCOBRL, caU 18064-014). Reac-tions were incubated at 40'C for 1h, diluted with H20 to 80 pi ands to re d a t -2 0'C .PCR ampli fi ca t ion

mRNA differential disp lay was performed essentially as de-scribed by Liang and Pardee (1992) with som e m odifications. Thefollowing c o m p o n e n t s w e r e m ix e d o n ic e in a P C R p la t e w ith 96w ells (Techne) or in 0.5 m l reaction tubes: 2111RT reaction, 2.5 pi10xPCR buffer (500 mM KCI, 100 mM Tris pH 9.0, 1% Triton X-100),2.5 pi MgCI2 (20 mM), 0.3 111[fl-"PI dATP (10 mCi/ml,DuPont NEN), 1 p15' primer (10 pM), 11113' prim er (10 pM ), 2.5pi dNTP mix (20 IlM dCTP, 20 pM dGTP, 20 I.M dTTP, 14 pMdA TP), 0.51.1 Am pliTaq@ DN A polym erase (5 U /pl, P erkin E lm er,cat.# N801-0060). The total volume was 25 pI. Reactions werek e p t o n ic e u n t i l t h e P C R m a c h in e h a d r e a c h e d 8 5C a nd th enapplied onto the PCR block, denatured at 92 'C for 2 min and thenamplified for 22, 30 or 38 cycles at 92'C for 50 see, 40'C for 90s e e , 7 2 ~ C f o r 6 0 s e c . A f t e r a m p l i f i c a t io n , r e a c t i o n s w e r e immedi-ately transferred to -20 'C .G el e lectrop horesis of D NA fragm ents

Three pi of each PCR reaction were m ixed with 1.5 1.1sam plebuffer (90% form am ide, 20 m M EDTA, 0.05% brom ophenol blue,0 .0 5 % x y le n e c y a n o le ) , d e n a tu r e d a t 8 0 ~ C f o r 4 m in , c h i l l e d o n ic ea n d lo a d e d o n to a 5 % d e n a tu r in g p o ly a c r y l a m id e (19:1a cry lamid e:b is ac ry lamid e) g el c on ta in in g 1 xT BE b uffe r. T o a ch ie vemaximal r e s o lu t i o n o f t h e c D N A b a n d s , t h e g e l w a s p r e r u n fo r 1 5m in in 0 .5 x T B E b u f f e r a n d , a f t e r l o a d in g , t h e s a m p le s w er e a l l o w e dto s e t t l e in t h e w e l l s f o r 5 m in b e fo r e r u n n in g th e g e l a t a c o n s t a n tpower of 45 W.D etection of cD NA band patte rns

Gels w er e t r a n s f e r r e d t o f i l t e r p a p e r , d r i e d a n d e x p o s e d to X - r a y 'f i lm o v e r n ig h t a t r o o m te m p e r a t u r e .Recovery and reamplification of DNA bands

W ith help of G logosTMIl Autorad M arkers (Stratagene, cat.#420202) the film was aligned with the gel and cDNA bands ofi n t e r e s t w e r e r e c o v e r e d b y c u t t i n g w i t h a r a z o r b la d e . T h e c u t t i n gn o rm a l ly in c lu d e d s o m e g e l a n d f i l t e r p a p e r , a n d t o a v o id c o n t a m i-n a t i o n b e tw e e n th e cDNA b a n d s s e l e c t e d , a n e w r a z o r b la d e w a su s e d fo r each band. The gel slice was transferred to a 1.5 m lr e a c t io n t u b e c o n t a in in g 4 0 0 ~ L I H 2 0 , b o i l e d f o r 1 5 m in a n d c e n t r i -f u g e d to s p in d o w n g e l a n d f i l t e r p a p e r . T h e s u p e r n a t a n t c o n t a in in gth e c D N A w a s t r a n s f e r r e d t o a n e w tu b e a n d th e D N A w a sp r e c i p i t a t e d with 10 pg glycogen. The pellet was dissolved in 20 piH20 and 10 ~I was used f o r r e a m p l i f i c a tio n in a r e a ct io n v o lu m e o f5 0 ~ I. T h e c o n d i t io n s w er e t h e s a m e a s b e f o r e , e x c e p t t h a t t h e f i n a ld N T P c o n c e n t r a t i o n w a s 4 0 p M in s t e a d o f 2 ~ L M a n d n o is o to p e w a sadded. One tenth of the reamplified cDNA was removed, andapplied to 2% a g a r o s e gel. The rema inde r o f t he reamplified cDNAwas p u r i f i e d u s in g W iz a r d P C R P r e p s D N A P u r i f ic a t i o n S y s t e m(P rome ga , c at.# A 71 70 ).


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Fig. 2. m RN A differential display using total R NA orpoly(A)+ RNA from male t cJ ) and fem ale ( 9 Iembryonic gonads, 13.5 dpc. Total R NA. DNasetreated total RNA and mRNA were prepared as de -sCr ib ed i nExp er im ent al P ro to co ls . (A I Th e 3 '-anchoredoligo(dT) pr imer 3 *4 was u se d f or re ve rs e t ra ns cr ip ti onand in com bination w ith the 5* 3 primer. cD NAs were amplified by PC R in the presence of la-33Pl dATP for3 0 c yc le s, r eso lv ed on a d en atu rin g p oly acry la mid e g el a nd ex po se d to X -ra y film B etw ee n 12.5 an d 1 00 n gtotal RNA per RT-PCR reaction w as used. The amount of poly(A )" RN A was more ddficult to determine. Ixc or re sp on ds t o a bo ut 25 ng , 2x to 50 ng and 4x to 10 0 ng total RNA per RT-PCR. Some RNA seems to haveb ee n lo st d urin g th e m RN A p urd lc atio n ste p. (8 ) mRNA d dfe re ntia l d isp la y u sin g d iffe re nt co mb in atio ns o f 3 'an d 5' prim ers. R everse transcription of poly(A )+ R NA from m ale and fem ale em bryonic gonads, 13 5 dpc,w ere p erfo rm ed b y u sin g tw o d iffe ren t o lig o(d T) p rim ers, 3* 2 an d 3*4. T he se , in c om bin atio n w ith 5 *2 , 5 *3an d 5 *20 w ere used for the subsequent PC R step, where cD NAs were ampldled for 22, 30 or 3 8c yc fe s. T hr eed iffe re ntia lly d isp la yed c DN A fra gm en ts a re in dic ate d w ith a rro ws, c DN A 1, 4 an d 80 Gels shown In (A andB) h av e b een e xp ose d o ve rn ig ht.

630 K. Nordql'isr alld I'. Tiihiill


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A lollse g01lad derelojJm ent alld m RNA d{fferelllial display 631

[a-"P] dATP (3000 Ci/m mol, DuP ont NEN ), 2 ~15' prim er (1 0 ~M ),2 ~13' prim er (1 0 ~M ), 0.5 ~I Am pliTaq@ DN A polym erase (5 U hll,Perkin Elm er). Sam ples were denatured for 2 m in at 92C and thenamplified for 5 cycles at 92 'C for 50 see, 40'C for 90 see, 72'C for60 sec. Probes were denatured by adding 100 ~II H20 to the PCRr e a c t i o n s a n d b o i l e d f o r 1 0 m in .H y b r i d i z a t i o n

F ilte rs w ere s oa ke d in 2 xSSC a nd p re hy brid iz ed in h yb rid iz atio nm ix (6xSSC, 3xDenhart's solution, 0.5% SDS) for 1 h at 60 'C .Denatured p r o b e s w e r e a d d e d a n d th e f i l t e r s incubated o v e r n i g h tat 60'C. Filters were washed 2x30 min in 2xSSC, 0.5% SDS at60'C and then exposed to X-ray film .DNA s eq ue nc in gDNA sequencing w a s carried o ut u sin g the D ye Term in ato rC yc le S eq ue nc in g R ea dy R ea ctio n k it (P erk in E lm er, c a1 .# 40 20 79 )an d the sa mp les w ere th en p ro cessed on a 3 73 A autom ated D NAs eq ue ncer ( Ap plie d B io sy st ems) .Semi-quantitative RT-PCRReverse t r a n s c r i p t i o n

Total RNA from 11.5 dpc, 12.5 dpc, 13.5 dpc and 17.5 dpcgonads+m esonephros and adult testis (0.16-4.5 ~g adult testisRNA for HPRT quantitative a nalysis and 0.5 ~ Ig m ale and fe malegonad +m esonep hro s R NA for 3 {3HSD I, HPRTand Srytimecourseanalysis) was m ixed with 2 ~I T,1NN primer m ix (TllCA, TllGC,TllCT, TllAG, 2.5 ~M of each) in a tota l volume of 14 ~I andi nc ub at ed a t 70:C f o r 8 m in . T h e fo l lo w in g c o m po n e n t s w e r e t h e nadded to each reaction: 5 ~tI5xRT buffer (200 mM Tris pH 7.5, 150mM K CI, 15 mM M gCI,), 2.5111DTT (0.1 M ), 2.5 ~I dN TPs (500 ~M ),0.5 ~ IIrR Nasin (40 U /~ II, P rom ega) an d 1.0 ~ I S upe rS cript II R T (200U/~I G IBCOBRL). Reactions were incubated at 45'C for 1 handt h en t r a ns f e rr e d t o -20C.S e m i - q u a n t i t a t i v e PCR

0.3 to 7.5 ~I (for HPRTquantitative a na ly sis ) o r 1 .5 11 1 (fo r Sry,H PR Tand 3 {3H SD lanalysis) of the R T reaction w ere m ixed on icein a PCR p la te w ith 9 6we lls (T ec hne) to ge th er w ith 2 .5 ~ 11 OxPCRbuffer (500 mM KCI, 100 mM Tris pH 9.0,1% Triton X-100), 2.5III 20 mM MgCI" 0.3 ~I [a-"P j dATP (3000 Ci/mmol, DuPontNEN), 1 ~I 5' primer (5'Sry or 5'3PHSDI or 5'HPRT, 100 ng/~I),1 ~13' primer (3'Sry or 3'3PHSDI or 3'HPRT, 100 ng/~I), 2.5 ~IdNTPs (500 ~M), 0.3 ~I AmpliTaq@ DNA polymerase (5 U/ltI,Perkin Elmer). The total volum e was 25 ~I. Reactions were kepto n ic e u ntil th e PCR machine had reached 85'C and then appliedonto the PCR block, denafured at 92'C for 2 min and thenamplified for 24 cycles at 92C for 60 see, 55'C for 60 see, 72'Cfor 60 sec. A ft e r t h e a m p l i f i c a t i o n , r e a c t io n s w er e im m e d ia t e lyt r a n s f e r r e d t o -20cC.Nativ e p oly ac ry lam id e g el e le ct ro ph or es is

Three ~I of each PCR were m ixed w ith 2 ~llloadin g buffe r (15 %F ic o l l , 0 . 2 % x y le n e c y a n o le , 5 0 m M E D T A ) a n d s e p a r a t e d o n a 5 %n a t i v e po lyacrylam ide (19:1 a crylam ide:bisacryla mide) gel con-taining 1 x T B E b u f f e r . G e ls w e r e t r a n s f e r r e d t o f i l t e r p a p e r , d rie dand eith er e xposed ove rnight to X -ray film or to P hosphorlm age rs c r e e n s fo r q u a n t i t a t i v e a n a l y s i s ( Im ag e Q ua n t s o f tw ar e ) .

Whole-mount i n s it u h y br id iz a t i o nD ig o x ig e n in - I a b e le d a n t i s e n s e a n d s e n s e R N A p r o b e s w e r e

p r e p a r e d f r o m l in e a r i z e d cDNA 4 . 3 . I n s i t u h y b r id i z a t i o n s w e r eperform ed as described by W ilkinson (1 992).

R es ults a nd D is cu ss io nH e r e w e p r e s e n t a s t r a t e g y f o r t h e is o l a t i o n o f g e n e s in d e v e lo p -

m en t a l b io lo g y s y s t e m s . T h is m e th o d in v o lv e s f o u r d i f f e r e n t s t e p s ;i ) a n m R N A d i f f e r e n t i a l d i s p la y m e th o d w h ic h w as o pt im iz ed f or u sein developmental biology, ii) a dot blot assay for the isolation o fp o s i t i v e cDNA c lo n e s , i i i ) a s e m i- q u a n t i t a t i v e R T .P C R a s s a y fo rr a p id a n a ly s is o f g e n e e x p r e s s io n , a n d iv ) w h o le -m ou n t in s it uh y b r i d i z a t i o n .

T h e p r o c e d u r e s h o w n in Figure 1 n ee ds less than 4 ~I g o f t ot alRNA, which can be obtained from two pairs of fetal gonads. Its h o u ld t h e r e f o r e b e p o s s ib le t o u s e th i s p r o c e d u r e f o r t h e i s o la t io na n d c h a r a c t e r i z a t i o n o f g e n e s in o th e r d e v e lo p m en t a l b io lo g ys y s t e m s w h e r e t h e a m ou n t o f R N A is l im i t e d .Optimization of conditions for m RNA differential display us-ing m inute am ounts of m aterial

Total RNA was isolated from male and female gonads withattached m esonephros (gonad+m esonephros) from 13.5 dpc oldmouse e m b r y o s . W e c h o s e t h is t im e p o in t f o r tw o r e a s o n s : f i r s t l y ,at 13.5 dpc the gonads have been developing for 1-2 days andgenes w h ic h a r e in v o lv e d in t h e s e p r o c e s s e s w i l l p r o b a b ly beexpressed (as for W I-I, Sf-I an d Sox-9). Secondly, it is easy tod is tin gu is h b et we en m a le a n d f e m a le gonads d u r in g th e d is s e c -t io n s , w h ic h means t h a t c o n t a m i n a tio n o f t h e m a le R N A p o p u la tio nb y f e m a le R N A s a n d v ic e v e r s a w i l l b e r e d u c e d to a m in im u m .

A s a f i r s t s t e p t h e a m o u n t o f t o t a l R N A r e q u ir e d f r o m m a le o rf e m a le 1 3 . 5 d p c g o n a d s + m e s o n e p h r o s was determ ined. It isshown in Figure 2A that as little as 12.5 ng of total RNA gave aproper and distinct cD NA banding pattern. 12.5 ng is equivalent tothe RNA from about 250 cells, based on published values of thetotal RNA in 8- to 16-cell and early b lastocyst stages, 40 pg/cell,and in regenerating rat liver, 60 pg/cell (P iko and Clegg, 1982;R e in e r s a n d B u s c h , 1 9 8 0 ) . I t i s p o s s ib l e t o u s e l e s s R N A , e v e n aslittle as 2 ng, but not all cDNA bands will be detected. This can bep a r t ia l l y o v e r c o m e b y lo n g e r e x p o s u r e t im es o f t h e g e l s .

L i a n g e t a l. h a v e s t r e s s e d th e im po r t a n c e o f D N a s e t re at m en t t oavoid DNA contamination in the PCR step (Liang et a l., 199 3).H o w e v e r , s in c e t h e D N a s e t r e a tm en t o f t e n r e s u l t s in lo s s o f R NAwe would like to om it this step. W hen total R NA and D Nase treatedtotal RNA were compared in the mRNA differentia l d isplay, nomajor d ifferences were detected in the banding pattern (F ig. 2A,lanes 1-2 and 9-10). A lso, when RT was performed on total RNAwithout r e v e r s e t r a n s c r ip t a s e , a lm os t n o b a n d s a p p e a r e d in t hemRNA differentia l display (data not show n). Therefore, by using am o d i f i e d v e r s io n o f t h e R N A e x t r a c t i o n m e th o d d e sc ri be d b yChomczynski and Sacchi (1987) which reduces the amount ofc on ta min atin g D NA , th e D Na se s t e p c o u ld b e o m it t e d .

W e also com pared total RNA with poly(A)" RNA and found thatthe banding pattern from poly(A)' RNA was cleaner. This m ade ite a s i e r t o d e t e c t c D N A b a n d d i f f e r e n c e s b e tw e e n m a le an dfem ale, particularly for cDN As longer than 300bp (Fig, 2A), It haspreviously been recommended not to use Poly(A)' RNA sinceo l i g o ( d T ) c o n t a m in a t i o n c o u ld g iv e a h ig h b a c k g r o u n d sm ear in


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632 K. Nordqri.H lIm l \'. Tiihalll'1l

- = ==.IJlJlU-I'(l(Qlllk:-';,\t P [)RT f '( "1 (cJrIlR~,\ - -t '" -= =~..hl...ul f he f '( l! : ,~~ "'lIh1I-.p.:P)J.-'TP

PAGEtCuI('(JII~D~AfnlglllC'nI.Rt:~"'rll~' fr~~nI:nIC1.flelnloTA-\t\:h..

P CH H f\ in j: lc : t "u lo oi cs t Jy u \i ng T A J 1I 1" J: "r \

Fig.3 . Selection of posit ive clones byd ot b lo t s cr ee ni ng . (A I A schema ti cp re sent at io n o f t he me th od , tB ) cDNA4( F i g 28 ) was e lu ted. r eampl lf ied andcloned mroa TA-vector . Smg le co lon ie swere picked. lysed and inserts weream plified by P CR and dot blotted ontotw o n ltr oc elf ul ose f,lt ers . Ma le (0 ' a ndfemale f 9 ) spe cIfic p ro be s w ere m adeb yexr en dmg the mRNA d ,f fe rent la ld is -p l a y (Fig 2 8 , lanes 3 an d 4) fo r 5 morecycles in the presence of {a-J1P ldA T Pas described In Experimental ProtocolsTwo inserts. 4.0 and 4.1. contained

cD NA s found In both male and female embryonic gonad. cDNA 4.3rep resen ted a n mRNA w hIc h IS ma le s pe cd lc In em bryo nic gon ad s

B ..... .. ...

the differential display (Liang et al., 1993). W e have not noticedt h i s problem in our differential display and are routinely usingPoly(A)' RNA as an extra safety step to avoid picking up falsep os itiv e c lo ne s c on ta in in g r RN A s eq ue nc es . H ow ev er, i f m i n u tea m o u n t s of materia lare u s e d fo r R NA ex tra ctio n it is a dvis able touse total RNA since poly(A) selection often result in loss of RNA.A lso, Zim merm ann and Schultz w ere successful i n u s i n g t o t a lR N A w h e n a n a ly z in g g e n e e xp re ss io n in th e p re im pla nta tio nm ouse em bryo by m A N A d iffe re ntia l d is pla y (Z im m er ma nn a ndSchultz, 1994). In any case, one of the most important factors fora successful mRNA differential display is to have RNA of goodquality and in equal amounts of the two RNA samples compared.

As suggested by Liang et at. (1 99 3), th e m eth od o f a nalyzingeach set of prim ers in duplicate w as adopted, i.e., tw o aliquotswere removed after the RT reaction and subjected to separatePCRs. Two different numbers of rounds of PCR cycles were used,2 2 c y c l e s t o detect d i f f e r e n c e s in t r a n s c r i p t s w h ic h a r e p r e s e n t inhigh trequency (Fig. 26, cDNA4) and 30 cycles for detectingdifferences in less abundant mRNAs (Fig. 26, cDNA 1).38 cycleswas also tested and gave the same result as for30 cycles (Fig. 2B,la ne s 5 a nd 6 ). S ep ar ate e xp erim en ts u s in g t h e s a m e p r im er p a i r

b ut o th er mRNA batc he s, d iffe re nt Ta q DNA polyme ra se s a ndo t h e r P C R m a c h in e s p r o d u c e d b a n d in g p a t t e r n s w h ic h w e r en e a r ly id e n t i c a l . H o w ev e r , s e p a r a t e w el l s in t h e P C R m a c h in ecould amp li fy d if fe ren tl y and i t i s impo rtan t to check the un iform it yo f th e we lls t o get r ep roducib le mRNA d iffe re nt ia l d is pla ys .T o a s s a y f o r d i f f e r e n c e s in m A N A pattern be tween male andfe ma le 1 3.5 dp c g ona ds+me so nep hros, a to ta l of 2 0 differe nta r b i t r a r y 5 ' - p r im e r s w e r e s c r e e n e d a g a in s t t h e fo u r d i f f e r e n t 3'-anchored oligo(dT) prim ers. Figure 26 displays three differentp r im e r c o m b in a t i o n s . a n d c l e a r l y s h o w s t h a t d i f f e r e n t p r im e r p a i r sgive different cD NA banding patterns, although the 3'-prim er w asthe same in lanes 1-6 and 11-14. On average, there was less thanone cD NA tragm ent w hich w as differentially displayed per pair ofp rim e r s . T h is seems r e a s o n a b l e s in c e L ia n g e t a l . ( 1 9 9 3 ) h a v er e p o r t e d t h a t le s s t h a n 1 % o f t h e c D N A b a n d s a p p e a r t o b ee x p re s s e d d if f e re n ti a l l y b etw e e n n o rm a l a n d b re a s t c a n ce r cells.Also, while Blanchard and Cousins (1996) found on average oned iffe re ntia lly d isp la ye d fra gm en t pe r d iffe re ntia l d isp la y re actio nwhen looking at intestinal mRNA regulated by dietary zinc.M o s t o f t h e t im e - c o n s u m in g w o r k r e m a in s a f t e r th e m R N Ad i f f e r e n t i a l d is p la y , i n c l u d in g v e r i f i c a t io n o f p o s i t iv e c lo n e s a n dd i f f e r e n c e s i n g e n e e x p r e s s io n . F o r t h is r e a s o n , a s e c o n d m RN Ad i f f e r e n t i a l d i s p l a y s c r e e n in g w a s p e r f o r m e d w i t h n e w R T r e a c -t i o n s t o e n s u r e t h a t t h e d i f f e r e n c e s in e x p r e s s i o n p a t t e r n s b e tw ee nthe male and female RNA populations were reliable (data notshown). After this, the cD NA bands of interest, cDNA 1,4 and 80,w ere cut out from the gel and ream plified.Iso latio n a nd s eq uen cin g of three d iffe re ntia lly d isp la ye dcDNAs

One of the m ajor drawbacks of the m RNA differential displaytechnique has been that the PCR products purified Irom gels inm any cases are heterogeneous (Bauer e t a l. . 1993 ; Ca ll ar d e t a l. ,1 994 ; L i e t a t. , 1 99 4; K . N o rd qvist, u np ub lishe d re su lts). T hu s, o necDNA band seen on the gel can contain cDNAs of both constitu-t iv e l y e x p r e s s e d a n d d i f f e r e n t i a l l y e x p r e s s e d g e n e s . S e v e r a la p p r o a c h e s h a v e b e e n u s e d t o c i r c u m v e n t t h is p r o b le m . T h e s einclude the direct use of the ream plified cD NA fragm ent as a probein Northern blot analysis, the use of Northern blot for affinitycapturing of cDNAs and separation of recovered cDNA bands bya single-strand conform ation polym orphism (SSC P) gel (Li e t a l.,1 994 ; Lia ng an d P ard ee, 19 95 ; M ath ie u-D au de e t a l. , 1 99 6; Z ha oe t a l. , 1996). However, the most commonly used procedure hasbeen to clone ream plified cD NA into suitable plasm id vectors andto use different dot blot m ethods to identity the clone of interest.D u ri ng t he la s t y e a r s a f lo r a o f p u b l ic a t i o n s h a s e m e r g e d w h ic hp r e s e n t s t e c h n ic a l im p r o v e m en t s w it h in t h i s f ie l d (C a l la r d e t a l.,1994; McClelland et al., 1994; Mou et al., 1994; Wong andMcClelland, 1994; Liu and Raghothama, 1996; Vogeli.Lange etat., 1 99 6; Z ha ng et at.. 1996). Most of these methods worke xce lle ntly fo r fin din g cD NA s w hich rep re se nt d iffe re ntia lly e x .p r e s s e d g e n e s in c e l l l in e s a n d l a r g e r a m ou n t o f t is s u e , b u t c a n n o tb e u s e d w i t h a l im i t e d a m o u n t o f m a t e r i a l , a s i s t h e c a s e indevelopmental biology. The advantage of the dot blot methodpresented here is that there is no need for extra RNA.

cDNA 1,4 and 80 were cloned into a plasm id vector (pCRTMII,Invitrogen) w ith help at the T A-cloning procedure which takesadvantage of the extra A nucleotide added by the Taq polym eraseto the 3'-end of the PCR fragments. To achieve high cloning


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Semi- quan tit ati ve RT-PCR assay s fo rde-termining mRNA levels of genes ex-p ressed d ur in g g on ad d evelo pm en t

In p re vio us a rtic le s d es crib in g th e mRNAd iffe re ntia l d is pla y te ch niq ue , N orth ern b lo tanalysis h a s b e e n comm only used to con-firm that a differentially displayed cDNAfragment represents a true difference intra ns crip t le ve ls b etw ee n th e s ample s te ste d(review ed by Liang and P ardee, 1995). It isu su ally imp os sib le to u se Nor th ern b lo t a na ly -s is whe n work in g in developmental biology,since the am ount of m ateria l is so sm all.

To confirm the differential expression ofmRNAs which correspond to cDNAs iso-lated by the mRNA differentia l d isplay, a semi-quantita tive RT-P CR assay w hich is sim ple, fast and w hich requires little RNA , w asdeveloped. Total RNA from female and male embryonicgonads+mesonephros w e r e reverse transcribed with a m ixture ofth e fo ur different 3'-anch ored o lig o(d T) prim ers used earlie r for themRN A diffe ren tial display. P CR p rim ers w ere selecte d so that theyw ould be located in t he 5' and 3' e nds of the cloned cD NA fra gm en t.Annealing was u su ally c arrie d out in the PCR at 55C>C,s in ce th ereaction w as successful at this temperature for m ost prim er pairstested. The PCR reaction w as optim ized to am plify only the targetsequence u n d e r non-saturated conditions, and was run for 24cycles in the presence of [a-"Pi dATP.

e ffic ie nc y, it w as im po rta nt to u se Amp liT aq(Perkin E lm er) in the ream plification step.P CR p rod ucts am plified w ith D NA P olym er-ase (DyNAZyme) or Taq DNA polymerase(Promega) and cloned into pCRT"11 gavelowe r c lo nin g e ff ic ie nc y.The principle of the modified dot blotassay that we developed is to make twoidentical n ylon filte rs, containing P CR am-plified inserts from single colonies. M alegonad specific and fem ale gonad specificprobes w ere m ade by am plifying an aliquotof the original differe ntial disp lay sam plesfor five additional cycles. This was per-form ed without reaching saturated condi-tions using the same set of primers asbefore in the presence of ra-"P] d AT P (F ig .3 A). T he re su lt from o ne d ot b lo t e xp erim en tis shown in Figure 36, where clone 4.3 wasshow n to be m ale sp ecific.T hre e d iffe re nt d ot b lo t e xp erim en ts w ereperform ed, one for each cDNA cloned, andpositive clones from each dot blot weresequenced (Fig. 4). cDNA 1.2 was 153bplong and not sim ilar to any D NA sequence sin the EM 8L database. cDN A 4.3 w as 255bplong and identical to the non-coding 3' endo f th e 3 ~-h yd ro xy ste ro id d eh yd ro ge na se lD5_D4 isomerase I (3~HSD I) gene. cDNA80.8 was 122bp long and homologous toa no th er s te ro id h yd ro xy la se g en e, GYP/T.

1.2

4.33(3HSDI4.33(3HSDJ4.3)(3HSDI

4.3)(3HSOI4.33(3HSDI

80.8C)p/780.8C)p/780.8C)p/7

MOllse gOliad dere/oplllell' alld IIIRNA dilferellIiai display 633

"2GCAAGTACCGGCAAGTACCGGGACAACCAGGGCTACACGGAGAAACCCTGTCTTCAAAACAAAAACAAACAAA TGAAAGTT TT CT TT CCTT CCT AT TGGTGCAA TCCA TACTTA T TCCAGGAAA T TCCT TAAGATGTATAGAIGTAAAATACAAAACTGTGCAAAAAAAAAAAGCTTTITTTTTTT"25.) 5.)(3I1SDICCA TOTCACCCCATGTCACCAGAAGCCTTGCCAGTCACTAACCCACTC AGAOAC CAAC TT GG T A T crrrC TC AAG T CACC AGAAGC CT TG CC AG TC AC T AAC CC AC T

CACAAACTTGCTTCCCTAAGCCCCTGCTCAGAGACAAACATAGCTGAGTCTCAGTTCITACACAAACTTGCTTCCCTAAGCCCCTGCTCAGAGACAAACATAGCIGAGTCTCAGTTCTTAGGCTTCAGCAATTACAATTTACTTTCACTTAGAACTTAGTATTGCTTTTATTTCCCCCTTGGCTTCAGCAATTACAA1.TACTTTCACTTAGAACTTAGTATTGCTTTTATTTCCCCCTT

3.)!3HSDICAAGTCCTAAATCATATCTTGCCTTTGGTAAAATCCCGTTGCTI .CTGAAGCAAAACAAACAAGTCCTAAATCATATCCTGCCTTTGGTAAAATCCCATTGCTTTCTGAAGCAAAACAAAAACAGCAATAAATATTGTAATGCCCTAAAAAAAAAAACAGCAA TAAATATTGTAA TGCCTT AAAAAAAAAGA TTTTTTTTTTTT

"413 J3 HSD c DNA 5 ' 1 6 2 1 1)'

1]67 1620


8/12

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'" ." ,, . '"'" '"F ig . 5 . Semi -qu an ti ta ti ve RT . PCR for th e analysis of 3{3HSD I an d Sr y expression. (A I C ontrol of PCR conditions, Different am ounts o f H PR T RNAf rom adu lt testis w ere reverse transcribed and introduced into P CR for 24 cycles. The PCR products w ere q ua ntita te d b y P ho sp ho r Im ag er a na ly sisR ea ct io ns w er e found to b e lin ea r between 30 and 270 ng RNA per RT-PCR. (B I 0 .5 1/ 9 t es ti s RNA w as re ve rs e tra ns crib ed a nd d iffe re nt amounts of theRT mix were applied to PCR. PCR fragments r e p r e s e n t in g H PR T transcripts were quantitated by Phosphor Im ager analysis. IC ) Tim e cou rse of 3f3HSDI e xp re ss io n. E xp re ss io n w as a na ly ze d u sin g s em i- qu an tita tlv e R T - P C R w ith the 3 f J H S O I s pe cd ic p rim ers d ep ic te d in F ig ure 4. T ota l R NA w as p re pa re das described In E xp erim en ta l P ro to co ls fro m 11.5 to 17 5 d p c f ema le (F ) a nd ma le (M! g on a ds+me so ne p hr os . 50 ng to ta l RNA was u se d in e ac h RT-PCR.ID ) T im e co urs e o f co ntro l. HPRT. The same RT reactions as fo r 3 f 3 H S O I expre ss ion were used IE } T ime cou rse o f Sr y exp ression. The same RNA asin (C an d 0) w as use d and reverse transcrib ed w ith a S ry specific 3' primer (3'"

S ry ). S ry e xp re ssio n le ve ls w ere q ua ntita te d b y P ho sp ho r Im ag er a na ly sis .

the PCR. The signal depended linearly on the amount usedbetween 10 and 150 ng of total R NA per RT-P CR . Forsubsequentanalyses, 500 ng total RNA was used in the RT reactions, and 1110 of the RT mix was used for each PCR. corresponding to 50 ngof total RNA per RT-PCR.

W ith help of the m RNA differential display m ethod presentedhere, three different cDNAs were isolated from the m ale gonadand sequenced, cDNA 1.2, cDNA 4.3 and cDNA 80.8. cDNA 1.2shows no homology with any known gene or DNA sequence.H owever, the difference in expression betw een m ale and fem ale13.5 dpc gonad+mesonephros was only approximately three

times. cDNA 4.3 and cDNA 80.8 showed almost perfect ho-m ology w ith ge nes w hich encod e steroid hydroxylases, 3{3HSDI and CYP17 respectively. cDNA 4.3 was chosen for furtheranalysis by sem i.quantitative RT-PC R and whole.m ount in s ituhybridization.

Two new primers, w ith a GC content of about 5 0 % , weresynthesized for assaying 3{3HSD I expression during gonaddevelopm ent (Fig. 4, 5 '3PHSDI and 3'3PHSDI). These prim erswere located close to the 5' and 3' ends of the cDNA 4.3 sequence,excluding t he AT -r ic h r e g i o n which is a t the very 3' end, and gaverise to a PCR fragm ent of 169bp. These prim ers were specific for


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~c,

i ..

...' . (,

Mouse gOliad del'eIoplllelll alld IIlRNA dif(l'rl'lliial display 635

.. ,.~,. .. .,

.

3t3HSD I and did not hybridize to transcripts from other 3pHSDgenes (data not show n).Gonads+mesonephros w e re r em o ve d f r o m e m br y o s o f P a r k e s

outbred m ice (NIMR) at different time points and total RNA wase xtr ac te d b y th e g ua nid in iu m is oth io cy an at e-a cid p he no l method.T he P ar kes m ic e w er e s ele cte d s in ce d et ai le d ti me c o u r s e s of Sryan d MIS e xp re ssio n from th is strain ha ve b een p ub lish ed (H ackeret al., 1995). The first time paint chosen was 11.5 dpc, 14 tails o m it e s , w h i c h is w h e n i t is f i r s t p os s i b le t o r emove the undif feren-t i a t e d g on ad +m e so ne ph ro s w ith ou t s ev er e c on ta m in ati on o f c el lsfrom the dorsal mesentery. In addition, the expression of Sry is am aximum at 11.5 dpc.

The semi-quantitative RT -PCR was used to com pare the timecourses of 3t3HSD 1 expression in fem ale and m ale developinggonads and to compare the onset of 3t3HSD I gene e xp re ss io nwith Sry expression, Figure 5C shows that very low levels of3/iHSD Im RN A w ere observed at 11.5 dpc, 14 tail so m ites, in bothmale and female gonad+mesonephros. Between 12.5 and 13.5dpc, 3t3HSD I expression increased dramatically in the malegonad+ mesonephros and at 17.5 dpc the level was about twentytimes higher than at 11.5 dpc. In contrast, female gonad+.

, .1I'.

.'Fig. 6. Expression of 3fJH SD I in fetal g onad sa nd a dr en al g la nd s. Whole-mount In S I r u hvbndl'zation was performed by using cDNA4.3 as DIG.labeled antisense probe. IA ) Fema le u ro geni ta lregion. 13.0dpcandlBJ 140dpc. showing3fJHSDf expreSSion in f e r a l adrenals. IC I Secr lon o f f e-male gonad, 1 4. 0 dpc. ID I M a le u rogen it al r eg ion ,13.0 dpc and lE I 14.0 dpc showing 3~HSO 1express/on m a dr ena ls a nd f er al tesris.IFI Seer/onof m ale gonad, 14.0 dpc, show ing 3{3HSD I e x-pre SS io n b etw ee n th e testis c ord s. IG ! 3{3HSD Ie xp re ss io n / n f e ta l a dr en al s, 12.0 to /4,0 dp c. (H )Secr/onof w hole-m oum show mg3{3HSD I expres-sion In correx cells o f r he f er al a dr en al , 14 0 d;x,a, adrenal; m, mesonephros: 0, fetal ovary, r. fetaltestis; t e. t es ti s c or d

mesonephros 3t3HSD 1 mRNA le ve ls sta ye d rela tive ly sta bleth ro ug ho ut th e tim e p erio d. HPRT e xp re ss io n was u se d to c he ckt ha t th e amount o f RNA was equal a t th e d iff er en t t ime poin ts ( Fig ,5D).3pHSD is a key enzym e in the biosynthesis at steroid hor-m ones.ln m ouse, five closely related genes w ith different expres-sion patterns have been charac te r ized, I to V (Bain e t a l.. 1991;Clarke e t a l., 1993 ; Abbaszade e t a l. . 1995). In the adult m ouse,3t3HSD I is the only 3pHSD gene which is expressed in bothgonads and adrenal glands. In fetal gonads+mesonephros wefound w eak 3t3HSD f expression as early as 11.5 dpc by semi.quantitative R T-PC R. The 3t3HSD I may be expressed in eithert he ind if fe ren t gonad or in the adrenal glands since the adrenalsa re n ot d et ac he d f r o m th e m es on ep hric tis su e u ntil 1 2.5 dpc, B yseparating the gonad from the mesonephros by d isse ctio n at11 .5 d pc it w ill b e p oss ib le to in ve stig ate th e so urce o f e arly3fJHSD I expressio n in m ore detail. The m ajo r increase in3/iH SD I exp re ss io n occur re d between 12.5 and 13.5 dpc. Leeand T aketa (1994) tound by R T-PCR that the expression of3t3HSD starts at 13.5 dpc during their investigation of sexre ve rs al in cro sses betw een C 57BL/6J an d B 6.y DOM , T hes e


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636 K. Nordqvi.H olld V. Tviliillell

results suggest that Leydig cells begin to differentiate between12.5 and 13.5 dpc in mouse.

The first sign of 3f3H SO I expre ssion in fem ale go nad occurredat 17.5 dpc, at which a slight increase in mRNA expression wasdetected by RT-PCR. In fetal and postnatal rat ovary, 3{3HSD ImRNA expression is first observed at day six after birth (Juneaue t a i, 1993). By using cDNA 4.3 as a probe, it w ill be possible tostudy m ore accurate ly the tim e cou rse of 3{3HSD lexpression an dto investigate the differentiation of steroid producing cells ind ev elo pin g o va ry .Sryencades two different transcripts in m ouse, a circular non-fu nc tio na l tra ns crip t w hic h i s expressed in the germ cells of adulttestis, and a functional transcript in t h e s o m at ic cells of theem bryonic m ale gonad (Capel e t a l., 1 99 3; Hac ke r e t a l., 1 99 5).PCR prim ers for analysis of Sr y exp re ss ion by semi -quan ti ta ti veRT-PCR were chosen such that they annealed only to the Sr ytranscript in the embryonic male gonad and not to the circulartranscript. It w as difficu lt to assay for Sr y expression w hen usingthe mixture of oligo(dT) prim ers in the RT step (data not shown),probably due to the presence of a 3400 nt long untranslatedregion at the 3' end and the low abundance of the Sr y transcript.For this reason, a Sr y specific 3' primer was used for RT.Sryexpression was detected in m ale embryonic gonad at 11.5dpc, which was the first time point exam ined, and decreased at12.5 dp c, consistent w ith previo us resu lts from RNase protectio nassays (Fig. 5E; Hacker e t a l. , 1995). Sr y is p ro ba bly e xp re ss edin pre-S ertoli cells since these ce lls are the first m ale specific cellsto differentiate in the teta l testis. In experiments w ith X X - X Vchim eric m ice, the S ertoli cell population is the only cell lineagewhich is alm ost exclusively com posed of XY cells, suggesting adirect action o f Sr y in Sertoli cells (Palm er and Burgoyne, 1991 a;Patek e t a l., 1991). The onset of 3{3H SD I expression was laterthan that of Sry, which agrees w ith the hypothesis that Leydig celldifferentiation is a consequence of Sertoli cell differentiation.

The semi-quantitative RT-PCR method presented here offersan easy way of looking at the expression pattern of genes whichare homologous to isolated, differentially displayed cDNAs. Whenthe amount of RNA needed has been titrated, this sem i-quantita-tive RT-PCR assay is easier to periorm than RNase protectionassay which needs highly labeled RNA probes and more totalRNA as starting m ateria l. W ith this sem i-quantitative RT-PCRa ss ay it is p os sib le to run one RT reaction with 500 ng total RNAand then to determine the expression pattern of about ten differentgenes,cDNA 4.3 is a useful m arker for steroid producing cells inem bryonic testis and adrenal glandLittle is known about the expression 01 3{3HSD I du ring ea rlytestis a nd o va ry de velopm ent in the mouse, I nor de r to invest igatethe localization of 3{3HSD I expression in fetal gonads and toconfirm the results from the sem i-quantitative RT-P CR, cD NA 4.3was used as an antisense probe in whole-mount in situhybridizations with urogen ita l tracts from 1 3,0 - a nd 1 4.0 -d ay -o ldembryos. No 3{3HSD I expression was detected in the gonadsfrom female embryos or in female or male mesonephros (Fig,6A,B,D). In contrast, the fetal adrenal glands of both sexes andthe fetal testis showed strong expression. In adrenal glands,3f3H SD I was expressed in the cortex region, which is the majorsite for steroid synthesis (Fig. 6G,H). In 13.0 dpc old testis, the

expression is patchy and m ainly concentrated in the middle andupper part of the gonad, less expression is found in the low er part(Fig. 60 ). T wenty four hours later, 3{3HSD le xp re ss io n is d et ec te din th e w ho le testis and restricted to h orizontal stripes of cells (Fig .6E ). S ections of the w hole-m oun t in s itu 14.0 dpc testis show edthat the striated appearance corresponded with expression be-tween testis cords, probably in the Leydig cells where testoster-one is produced (Fig. 6F). In sum mary, the whole-m ount in s ituhybrid izations showed that cDNA 4.3 is a usefu l marker for thed iff er en tia tio n a nd organization o f L ey dig c ells d urin g te stis d ev el-opment. 3{3HSD I is expressed in the male but not the femalegonad at 13.5 dpc, w hich is consistent with results from the m RNAdifferential disp lay an d sem i-quantitative R T-P CR . Little is kno wnabout early Leydig cell m igration and differentiation and it w ill beinteresting to analyze the three-dimensional organization of Ley-dig cells during testis cord formation. This could be done usingconfocal m icroscopy with cD NA 4.3 as a D IG -labeled anti-senseprobe and a fluorescing antibody towards DIG.AcknowledgmentsT his work was initiated at the Laboratory o f D evelo pm ental G en etics,Med ical Research Council, Na tional Institu te for Medical Research,London . W e thank Dr, R obin Lovell-Badge fo r h is support and excellen tscien tific g uidance, D r. K arin S ch me kel an d D r. C hrister H aa g for critica lreading of the manuscript. This work was supported by the SwedishN atura l S cie nce Found atio n, Erik Philip-S6 rensens Fo unda tion , M agn.Bergva lls Foundation, Jeanssons Founda tion and the Karo finska Insti-tute.

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mrna display

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