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DEVELOPMENTAL BIOLOGY 180, 499–510 (1996)ARTICLE NO. 0323

A Receptor Tyrosine Kinase, Sky, andIts Ligand Gas 6 Are Expressed in Gonads andSupport Primordial Germ Cell Growth orSurvival in Culture

Nobumichi Matsubara,* Yoshihiko Takahashi,* Yukio Nishina,†Yousuke Mukouyama,‡ Masahiro Yanagisawa,* Toshio Watanabe,‡Toru Nakano,§ Koji Nomura,§ Hitoshi Arita,§ Yoshitake Nishimune,†Masuo Obinata,* and Yasuhisa Matsui*,1

*Department of Cell Biology, Institute of Development, Aging, and Cancer, Tohoku University,4-1 Seiryo-machi, Sendai 980, Japan; †Department of Science for Laboratory AnimalExperimentation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka,Osaka 565, Japan; ‡Department of Molecular Information, Institute of Molecular and CellularBiosciences, Tokyo University, 1-1 Yayoi, Tokyo 113, Japan; and §Shionogi ResearchLaboratories, Shionogi & Co., Ltd., Osaka 553, Japan

Although a number of growth factors and their receptors are involved in the proliferation and differentiation of primordialgerm cells (PGCs), the only factor that has been shown to be active in vivo is Steel factor, a ligand for c-Kit. To identifynew growth factor receptors that may be required for PGCs function in vivo, we used an reverse transcription–polymerasechain reaction-based strategy to screen for protein kinase genes expressed in PGC-derived embryonic germ cells. We reporthere that one such gene encoding the receptor tyrosine kinase, Sky, is expressed in both PGCs and their supporting cellsin male genital ridges after 11.5 dpc. Interestingly, Sky expression was not detected in female genital ridges, althoughtranscripts were detected in supporting cells in the developing ovary at later stages. Gas 6, a ligand for Sky, was alsoexpressed in interstitial cells which surround Sky positive cells in genital ridges, and, in addition, it supported PGC growthor survival in culture. After birth, Sky expression in testis was restricted to Sertoli cells, and Gas 6 was detected aroundperitubular cells and Leydig cells. These results suggest that Gas 6–Sky signaling plays a role in PGC growth, sexualdifferentiation, and Sertoli cell functions in vivo. Sky expression in Sertoli cells diminished by 3 weeks of age, whenhaploid germ cells first appear. On the other hand, the expression in Sertoli cells was markedly upregulated in the testisof germ cell-deficient W/Wv and jsd/jsd mice. The results suggest that signals from differentiated germ cells suppress Skygene expression in Sertoli cells. High-resolution chromosomal mapping of Sky is also reported. q 1996 Academic Press, Inc.

INTRODUCTION matogonia (Yoshinaga et al., 1991; Rossi et al., 1993; Tajimaet al., 1994) and for the maintenance of growing oocytes(Packer et al., 1994). Other growth factors, including leuke-The proliferation and differentiation of germ cells are reg-mia inhibitory factor (LIF) (De Felici and Dolci, 1991; Mat-ulated by a number of environmental and intrinsic factors.sui et al., 1991), basic fibroblast growth factor (bFGF), (Mat-One of the most important is Steel factor, which is neces-sui et al., 1992; Resnick et al., 1992), and tumor necrosissary not only for the proliferation and survival of primordialfactor a, (TNFa), (Kawase et al., 1994), synergistically pro-germ cells (PGCs) (Godin et al., 1991; Dolci et al., 1991;mote PGC growth with each other in vitro. Activin A andMatsui et al., 1991), but also for the development of sper-IL-1a are also known to stimulate the growth of spermato-gonia (Jegou, 1993). It is, however, not clear whether thesefactors affect germ cells in vivo. Although PGCs express1 To whom correspondence should be addressed: Fax: 81-22-717-

8488. E-mail: [email protected]. LIF receptor (LIFR) on their surface (Cheng et al., 1994),

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0012-1606/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

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500 Matsubara et al.

mice lacking the LIF gene (Stewart et al., 1992) or the low- and play a role in PGC function, we have attempted toisolate such genes from a PGC-derived embryonic germ (EG)affinity LIFR gene (Ware et al., 1995) are fertile. OSM, a

factor that shares the LIFR/gp130 complex as a specific re- cell line. One of these genes was specifically expressed ingonads and brain in the adult mouse and was identical toceptor, also stimulates PGC growth in vitro (Cheng et al.,

1994) and may be physiologically active in vivo. More re- Sky previously cloned from a hepatoma cell line, brain, anda teratocarcinoma cell line (Ohashi et al., 1994; Mark et al.,cently the physiological importance of soluble factors Des-

ret hedgehog (Bitgood et al., 1996) and BMP8b (Zhao et al., 1994; Lai et al., 1994; Crosier et al., 1994). Sky encodes areceptor tyrosine kinase, which has immunoglobulin-like1996), which are secreted from Sertoli cells and spermato-

cytes, respectively, has been reported. Null mutants for and fibronectin type III-like repeats in its extracellular do-main that are characteristic of neural cell adhesion mole-these genes exhibit male germ cell deficiency indicating

that they are positive regulators for male germ cell growth, cules. It has recently been reported that Gas 6, originallycloned from serum-starved NIH3T3 cells (Schneider et al.,survival, or differentiation in vivo.

Direct interaction between germ cells and their support- 1988; Manfioletti et al., 1993), was a ligand for Sky. Thestructural feature as a cell adhesion molecule and the spe-ing cells is necessary for normal gonad development. Steel

factor may be synthesized as either a soluble or membrane- cific binding with the ligand suggest that Sky could functionthrough homophilic interaction as well as binding with Gasassociated form. The importance of the latter in vivo has

been shown by analysis of homozygous Sld mutant mice 6 (Godowski et al., 1995; Ohashi et al., 1995). We reporthere that Sky and Gas 6 are expressed in developing gonadswhich produce only the soluble form due to a structural

alteration of the gene. Development of germ cells is im- and that recombinant Gas 6 can support PGC growth orsurvival in culture.paired in these mice (Flanagan et al., 1991; Brannan et al.,

1991). A role of supporting cells for development of PGCsis also suggested by mice homozygous for a disruption ofthe Ftz-F1 gene, which encodes an orphan nuclear receptor. MATERIALS AND METHODSFtz-F1 is expressed in pre-Sertoli cells and interstitial cellsin the genital ridges (Luo et al., 1994), and in the Ftz-F1 null Isolation and sequencing of murine GCK4 (Sky) cDNA. A frag-embryos, cells in genital ridges undergo apoptosis, causing ment of murine GCK4 (Sky) cDNA was obtained from EG cells by

an reverse transcription-polymerase chain reaction-based strategydegeneration of the developing gonads. PGCs normally col-as described (Matsubara et al., 1995). To obtain longer cDNAs, aonize the developing genital ridges by 11.5 dpc in the mu-murine adult Balb/c brain cDNA library (Stratagene) was screenedtant, but markedly diminished numbers of PGCs are foundusing the PCR clone as a probe, and 20 overlapping clones wereat later stages. Although precise functions of Ftz-F1 in de-obtained. The longest had a 3.3-kb insert and contained most ofveloping genital ridges are not well defined, the results sug-the coding region and 3* untranslated region. The 5* region of Skygest that the failure of PGC growth is caused by impropercDNA was obtained by 5*-RACE. The entire nucleotide sequences

development of the supporting cells. On the other hand, of the clones were determined using Pharmacia ALF sequencer,germ cells are thought to be necessary for supporting cells. and the nucleotide sequence and deduced amino acid sequencesFor example, in the absence of germ cells Sertoli cells fail were analyzed with GenBank and EMBL data bases.to produce a number of important proteins (Jegou, 1993). It In situ hybridization. In situ hybridization using a digoxigenin

(DIG)-labeled RNA probe was carried out essentially as describedis therefore of interest to identify molecules involved in(Hirota et al., 1992). All embryos and gonads except WBB6F1-W/germ cell-supporting cell interaction.Wv testis were obtained from Balb/c mice and were dissected ac-The importance of receptor tyrosine kinases (RTKs) andcording to Hogan et al. (1995). Tissues were fixed in 4% paraformal-their ligands in cell–cell communication during develop-dehyde and embedded in paraffin. Five-micrometer sections werement has been amply demonstrated. For example, c-Kit anddewaxed, rehydrated, and pretreated with 20 mg/ml proteinase Kits ligand are essential for the proliferation, survival, and/for 8 min, with 0.2 M HCl for 10 min and with 0.25% acetic anhy-

or differentiation of hematopoietic cells and melanoblasts, dride in 0.1 M triethanolamine for 10 min. The dehydrated sectionsas well as germ cells (Williams et al., 1992). Other examples were then hybridized at 507C for 16 h in 50% formamide, 10 mMare fibroblast growth factors (FGFs) and their receptors, Tris/HCl, pH 7.6, 200 mg/ml yeast tRNA, 11 Denhardt’s, 10%which are involved in development of the postimplantation dextran sulfate, 600 mM NaCl, 0.25% SDS, and 1 mM EDTA. The

slides were then washed at 657C for 30 min in 50% formamide,embryo (Feldman et al., 1995; Yamaguchi et al., 1994; Deng21 SSC, and treated with 10 mg/ml RNaseA at 377C for 30 min,et al., 1994), and platelet-derived growth factor (PDGF) re-followed by washes in 21 SSC and 0.21 SSC at 507C. After washing,ceptor a, which plays a critical role in the differentiationslides were incubated with anti-DIG–alkaline phosphatase conju-of mesodermal and neural crest-derived mesenchyme (Ste-gate for 30 min and then reacted with 4-nitroblue tetrazolium chlo-phenson et al., 1991; Schatteman et al., 1992). Members ofride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate, 4-toluidinethe Trk receptor family and their ligand neurotrophins aresalt (BCIP). The sections were counterstained with methyl green.

also major regulators of nervous system development The template DNA for in vitro RNA synthesis was a 967-bp SacI(Davis, 1994). Taken together, RTKs have emerged as im- fragment of Sky cDNA that covers a part of coding region.portant molecules that regulate cell type specification dur- Immunohistochemistry. For immunohistochemistry, rabbiting development. anti-rat Gas 6 polyclonal antibody which was raised against the

purified rat Gas 6 was used. Cryosections of tissues from Balb/cTo identify receptor tyrosine kinases that are expressed

Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.


501Sky and Gas 6 in Developing Gonads

mice and embryos were fixed in PLP fixative and were treated in turing polyacrylamide gel and photographed after staining withethidium bromide.peroxoblock (Zymed). Sections were stained using a Histostain SP

Kit (Zymed) according to the instruction of the manufacturer andwere counterstained with hematoxylin. The concentration of theprimary antibody was 5 mg/ml. RESULTSPrimary culture of PGCs. Primary culture of murine PGCs wascarried out as described (Matsui et al., 1991, 1992). The caudal

Cloning and Structural Analysis of GCK4 (Sky)region of 8.5 dpc embryos obtained from ICR1 B6D2F1 mating wasdissociated into single cells by trypsin treatment. Cells from the cDNAequivalent of 0.5 embryos were seeded into a well containing Sl/

To isolate cDNAs encoding receptor tyrosine kinases thatSl4-m220 cells or Sl/Sl4 cells as feeder layers (Matsui et al., 1991,play a role in germ cell development, we employed a PCR-1992). Sl/Sl4 is a stromal cell line derived from the hematopoieticbased method using RNA obtained from EG cells and prim-microenvironment of a homozygous Sl/Sl murine embryo in whichers corresponding to a conserved region in the kinase do-the entire coding sequence of Steel factor gene is deleted (Toksoz

et al., 1992). Sl/Sl4-m220 is a derivative of Sl/Sl4, which expresses main as described (Matsubara et al., 1995). One of the clonesonly the alternatively spliced form of mouse Steel factor in which (GCK4) was found to be predominantly expressed in thethe extracellular proteolytic cleavage site encoded by exon 6 is testis, ovary, and brain in the adult mouse by Northern blotmissing (Matsui et al., 1991). Rat recombinant Gas 6 was added at hybridization (data not shown). We next screened a braina concentration of 350 ng/ml, which was shown to be optimal for cDNA library with the PCR fragment of GCK4, and thePGC growth.

longest 3.3-kb cDNA and 5*-RACE clones were sequenced.Northern blot hybridization. Total RNAs were prepared fromConsequently, we found that GCK4 was identical with pre-testes of postnatal ICR mice, 3- to 4-month-old WBB6F1-W/Wv

viously isolated cDNAs designated human Sky (Ohashi etmice, and 4.5-month-old B6-jsd/jsd mice by acid guanidium thiocy-al., 1994), human and mouse rse (Mark et al., 1994), mouseanate–phenol–chloroform (AGPC) method (Chomczynski and Sac-Tyro3 (Lai et al., 1994), and human and mouse DTK (Croiserchi, 1987). The germ cell-rich fraction and Leydig cell-rich and

tubular cell-rich fractions were obtained from 3- to 4-month-old et al., 1994) and was very closely related to mouse brt (Fuji-B6 testes and 3- to 5-month-old jsd/jsd testes, respectively. Testes moto and Yamamoto, 1994) and human tif (Dai et al., 1994).were treated with 0.1% collagenase and fractionated by centrifuga- The cDNA encodes a receptor tyrosine kinase that hastion as described (Tung et al., 1984; Maekawa and Nishimune, structural similarity to Axl/Ufo/Ark (O’Bryan et al., 1991;1985). Thirty micrograms of each RNA was separated by 1% aga- Faust et al., 1992).rose/Mops– formaldehyde gel electrophoresis, and Northern blothybridization was carried out under high stringency conditions ac-cording to Sambrook et al. (1989). A 32P-labeled 967-bp SacI frag- Sky Is Differentially Expressed in Male and Femalement of Sky cDNA was used for a probe. Embryonic and Neonatal Gonads

Interspecific backcross mapping. The large backcrossWe first examined the distribution of Sky transcripts inpanel between the two mouse species Mus spretus and

embryonic and neonatal gonads by in situ hybridization. AtC57BL/6 containing 1000 animals was generated jointly by10.5 dpc, PGCs migrate through the dorsal mesentery to-the Human Genome Project Resource Center, UK, and theward the developing genital ridges. Some cells along thePasteur Institut, France. (C57BL/6 1 M. spretus) F1 femalesdorsal mesentery and around the dorsal aorta showed weakwere backcrossed to C57BL/6 or M. spretus males. Eachsignal (data not shown). At 11.5 dpc, no significant signalbackcross progeny mouse has been scored for three to fourabove background was observed in both male and femalemicrosatellite markers per chromosome, completing an an-genital ridges (data not shown).chor map of 70 loci across the mouse genome. From 873

Abundant expression of Sky transcripts was seen in 12.5backcross progeny, a total 62 animals, which are recombi-and 14.5 dpc male genital ridges (Figs. 1A–1C). The signalnants between D2Mit11 and D2Nds3 on chromosome 2,was distributed in both PGCs, identified by their relativelywere used to determine the precise chromosomal locationlarge and round nuclei, and adjacent pre-Sertoli cells. Noof Sky.signal was found outside of the testicular cords where inter-Southern blot hybridization and PCR amplification. DNA (5

mg) was digested with appropriate restriction enzymes, separated stitial cells and Leydig cells are found. In contrast, Sky tran-through 0.7% agarose gel, and blotted to Hybond N/ membranes scripts were not detected in female genital ridges at 12.5(Amersham) by standard procedures (Sambrook et al., 1989). A ra- and 14.5 dpc (Fig. 1G and data not shown). Signal on meso-diolabeled Sky cDNA fragment (2.2-kb BglII–HincII fragment) was nephros was also seen using a sense probe and seemed toprepared using a random primer kit (Boehringer-Mannheim). The be nonspecific (data not shown).assay for detection of microsattelite markers (Dietrich et al., 1994) At 17.5 dpc, the testis cords are filled with gonocytes,included a denaturation step at 947C for 4 min followed by 30

recognizable by their round nuclei. They were positive forcycles of 947C for 1 min, 557C for 1 min, 727C for 1 min, and a finalSky expression, although the signal was weak. Stronger sig-elongation step at 727C for 7 min. Taq polymerase (Amersham) wasnal was seen around the nuclei of Sertoli cells that are pe-used under assay conditions specified by the manufacturer. Theripherally located in the testis cords (Fig. 1D). At birth, Skyconcentration of each primer was 0.7 mM. Microsatellite markertranscripts were detected in the center of the cord thatprimer sets (Map Pairs) were purchased from Research Genetics

(Huntsville, AL). PCR products were separated on a 8% nondena- mainly contains Sertoli cell cytoplasm. Gonocytes that



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have large and round nuclei in the cord were negative (data 1995). In contrast, Gas 6 showed no effect on PGCs culturedon the feeder layer of Steel factor-lacking Sl/Sl4 cellsnot shown).

In the developing ovary, some interstitial cells in the cen- (Fig. 3B).tral region showed signal above background at 17.5 dpc,but peripherally located oogonia were negative (Fig. 1H). At

Sky Is Specifically Expressed in Sertoli Cells inbirth, cuboidal cells surrounding follicles that had initiatedPostnatal Testes, While Differentiated Testiculargrowth were positive in the central part of the ovaries (Fig.Germ Cells Suppress Its Expression1I), but most of the flattened follicle cells in primordial

follicles and oocytes were negative for Sky expression. After By 7 days of age, most spermatogonia migrate to the pe-riphery of the testis cords, and the central region where1 week of age, no localized signal was found in the ovaries

(data not shown). Sky expression was prominent only contains Sertoli cellcytoplasm (data not shown). Little or no expression wasseen around the nuclei of spermatogonia. At 21 days after

Gas 6 Protein Is Expressed in Genital Ridges and It birth, when haploid cells first appear, signal was restrictedSupports PGC Growth or Survival in Culture around the basally located nuclei of Sertoli cells in all semi-

niferous tubules, and signal was markedly diminished (dataWe next examined the expression of Gas 6, a ligand forSky, in murine gonads by immunohistochemistry with not shown). The same pattern of expression was maintained

from 35 days of age onward (Fig. 1E). To determine the timeanti-Gas 6 antibody. At 11.5 dpc no significant signal abovebackground was observed in both male and female genital course of expression of Sky transcripts in developing testes,

we prepared RNA from postnatal testes and carried outridges (Figs. 2A and 2G). Strong staining was seen in intersti-tial cells which were surrounding Sky-expressing cells in Northern blot hybridization. In agreement with the results

of in situ hybridization, the level of Sky transcripts gradu-male genital ridges at 12.5 and 14.5 dpc (Figs. 2B–2D).Weaker signal was also detected in female genial ridges ally diminished after 10 days of age (Fig. 4). Sertoli cell-

specific expression of Sky in adult testis was confirmed by(Figs. 2H and 2I). In adults, Gas 6 existed in peritubular cellsand endothelial cells in testes (Figs. 2E and 2F) and in theca Northern blot hybridization using RNA prepared from frac-

tionated testicular cells. Significant expression was seen incells and corpus luteum in ovaries (data not shown).The expression of both the receptor and the ligand in the tubule fraction that contains Sertoli cells exclusively,

while only faint signal was found in germ cell and Leydiggenital ridges suggests that they are involved in PGC growthor survival. To investigate this, we tested the effect of re- cell fractions (Fig. 4). A higher level of Sky expression was

detected in the germ cell-deficient testis of W/Wv mice (Fig.combinant Gas 6 on PGC growth in culture. We used PGCsobtained from 8.5 dpc embryos, which show similar respon- 4). This was not due to the difference in Sertoli cell number

between the mutant and normal mice, because expressionsibility to various growth factors as gonadal PGCs (Matsuiet al., 1991, 1992; Godin et al., 1991; De Felici and Dolci, in each Sertoli cell in the mutant testis was stronger than

that detected in normal testis by in situ hybridization (Fig.1991; De Felici et al., 1993). PGCs increased in number onthe feeder layer of Sl/Sl4-m220 cells expressing membrane- 1F). Upregulated expression of Sky in testes was also ob-

served in jsd/jsd (juvenile spermatogonial depletion) miceassociated Steel factor without addition of Gas 6, but thenumber of PGCs was significantly higher in the presence by Northern blot (Fig. 4). In jsd/jsd mice, the first wave of

spermatogenesis occurs, but only type A spermatogonia andof Gas 6 than that in controls at 4 days in culture (Fig. 3A),and the increase was dose-dependent with an optimum at Sertoli cells are found in the seminiferous tubules after the

juvenile stage due to the inability of type A spermatogonia350 ng/ml (Fig. 3C). The optimum concentration was simi-lar to that for vascular smooth muscle cells (Nakano et al., to differentiate (Mizunuma et al., 1992).

FIG. 1. Expression of Sky mRNA in embryonic and postnatal gonads. Sagittal sections of male genital ridges at 12.5 dpc (A) and 14.5dpc (B, C), testes at 17.5 dpc (D) and 35 days after birth (E), testis from W/Wv mouse at 8 weeks of age (F), female genital ridges at 14.5dpc (G), and ovaries at 17.5 dpc (H) and 2 days after birth (I) were hybridized with DIG-labeled antisense RNA probe specific for mouseSky. Sky was expressed in the cells in forming testicular cords (t), whereas no significant signal above background was found in femalegenital ridges (A–C, G). Signal on mesonephros was nonspecific. Positive signal was also seen around presumptive gonocytes (gc) andSertoli cells (s) in the testicular cords at 17.5 dpc (D). Peripherally located Sertoli cells gave weak signal at 35 days (E), whereas muchstronger signal was found in Sertoli cells in W/Wv testis (F). In ovaries, weak but significant signal above background was seen in interstitialcells (i) in the central region and in cuboidal cells (c) (H, I). Signal around rete and outside ovary was nonspecific. gr, genital ridge; m,mesonephros. Bar: (A, B, G) 100 mm, (E, F, H) 50 mm, and (C, D, I) 20 mm.FIG. 2. Expression of Gas 6 in gonads. A transverse section of male (A) and female (G) embryos at 11.5 dpc, sagittal sections of male (B–D) and female (H, I) genital ridges at 12.5 dpc (B, H) and 14.5 dpc (C, D, I), and sections of testes at 14 weeks of age (E, F) were stainedwith anti-Gas 6 antibody. Positive signal was seen around interstitial cells (i) in genital ridges (B–D, H, I) and around peritubular cells(pt) and endothelial cells (et) in testis (E, F). No significant signal was observed in genital ridges at 11.5 dpc (A, G). da, dorsal aorta; me,mesentery. Bars: (A, G) 100 mm, (C, E) 50 mm, and (B, D, F, H, I) 20 mm.




FIG. 3. Effect of recombinant rat Gas 6 on 8.5 dpc PGC growth in culture. PGCs from 8.5 dpc embryos were cultured with membrane-associated Steel factor-expressing Sl/Sl4-m220 feeder cells (A) or Steel factor-lacking Sl/Sl4 feeder cells (B) either with (solid bars) or without(open bars) 350 ng/ml Gas 6. The effect of different concentrations of Gas6 was tested on Sl/Sl4-m220 feeder cells (C). Cultures were fixedand the number of alkaline phosphatase positive cells was counted on Day 1 and Day 4 in culture. Each experiment was carried out withduplicate wells, and numbers are the means { SEM of eight (A), six (B), and one (C) separate experiment. *Significantly different fromcontrol at P õ 0.05 by Student’s t test.

Fine Chromosomal Mapping of the Sky Gene precisely, we selected a total of 62 of 187 recombinantsbetween D2Mit11 and D2Nds3 from 873 backcross progeny.To investigate whether the Sky gene is localized to aThese recombinants were further typed for D2Mit42,region of the mouse genome involved in gonad develop-D2Mit103, D2Mit17, D2Mit190, Sky, and D2Mit164. Thement, fine chromosomal mapping was carried out using angenetic order of this region is determined by the haplotypeinterspecific backcross panel.analysis shown in Fig. 5A. The genetic order and distancesC57BL/6 and M. spretus genomic DNAs were digested({1 standard error) are as follows: Cen- - -D2Mit11-11.77 {with several different restriction enzymes and analyzed by1.90 cM-D2Mit42-4.84 { 1.26 cM-D2Mit103-0.35 { 0.35Southern blot hybridization for restriction fragment lengthcM-D2Mit17-0.35 { 0.35 cM-Sky/D2Mit190-0.69 { 0.49polymorphisms (RFLPs). An informative RFLP was obtainedcM-D2Mit164-3.56 { 1.08 cM-D2Nsd3. A linkage map offollowing digestion with BglII, yielding a 6.4-kb hybridizingthe loci included in this study on chromosome 2 is shownband in M. spretus and a 4.4-kb band in C57BL/6 (data notin Fig. 5B.shown). PCR analysis was also made on both DNAs to find

As discussed below, several uncloned mouse mutants ex-microsatellite polymorphisms. Both C57BL/6- and M.ist around Sky, but none of these mutations has a phenotypespretus-specific RFLPs and sequence variations were fol-associated with abnormalities in gonad development.lowed in backcross mice. Initially, we analyzed 47 randomly

picked backcross DNAs and obtained the initial assessment DISCUSSIONof the linkage relationship of the Sky gene. This analysismapped Sky to mouse chromosome 2 between D2Mit11 and We have cloned a cDNA encoding Sky receptor tyrosine

kinase from EG cells and have found that the gene wasD2Nds3. To reveal the chromosomal location of Sky more




FIG. 4. Detection of Sky mRNA by Northern blot hybridization in developing and germ cell-deficient testes and fractionated testicularcells. Each lane contained 30 mg of total RNA. Lanes 1–10, total RNA from 0- to 4-, 6-, 8-, 10-, 12-, 14-, 16-, 18-, and 24-day-old and 2-to 3-month-old testes; lane 11, 3- to 4-month-old W/Wv testis; lane 12, 4.5-month-old jsd/jsd testis; lane 13, germ cell fraction; lane 14,tubule fraction; and lane 15, Leydig cell fraction. The positions of ribosomal RNA are shown. The blots were striped off and reprobedwith the b-actin probe as an internal control.

expressed in PGCs, supporting cells in male genital ridges pathway, and can bind to Sky (Godowski et al., 1995;Ohashi et al., 1995). Our results (Figs. 1 and 2) suggest thatafter 11.5 dpc (when Sertoli cell differentiation starts), and

in prenatal testes, but the expression was not detected in Gas 6 produced in interstitial cells in genital ridges andadult testes diffuses in the extracellular space to interactfemale genital ridges by in situ hybridization (Fig. 1). We

have also found that Gas 6, a ligand for Sky, was expressed with Sky expressed on PGC and Sertoli cell surfaces to sup-port their growth and/or functions. Although the recombi-in both male and female genital ridges after 11.5 dpc (Fig.

2) and that the recombinant Gas 6 supported PGC growth nant Gas 6 supported PGC growth or survival on mem-brane-bound Steel factor-expressing Sl/Sl4-m220 cells inor survival in primary culture (Fig. 3). At later stages, Sky

is expressed only in supporting cells, i.e., Sertoli cells in culture (Fig. 3A), it had no effect on PGCs cultured on Sl/Sl4 cells which lacked the Steel factor gene (Fig. 3B). Thistestes and granulosa cells in ovaries (Figs. 1 and 4), and Gas

6 was expressed in peritubular cells in testis (Fig. 2). We indicates that Gas 6 enhances Steel factor-induced growthor survival of PGCs. A similar effect of Gas 6 has been foundconcluded that the expression of Sky in Sertoli cells was

suppressed by differentiated testicular germ cells for the on vascular smooth muscle cells, but in this case, Gas 6enhances proliferation of the cells, which is induced byfollowing reasons: (1) The expression in developing testes

was diminished after 10 days of age, when spermatogenesis growth factors activating Ca2/-mobilizing receptors (Na-kano et al., 1995).begins (Figs. 1 and 4). (2) Sky expression in Sertoli cells was

markedly upregulated in testes of germ cell-deficient W/Wv From the structural feature of Sky, there is another possi-ble model for Sky function. The extracellular domain of Skyand jsd/jsd mice whose spermatogonia fail to develop (Figs.

1 and 4). Taken together, our findings suggest that Gas 6– is composed of two immunogloblin-like domains and twofibronectin type III domains that are similar to the extracel-Sky signaling contributes to gonad development in many

aspects. lular domains of receptor tyrosine kinase, Axl/UFO/Arkand Eyk (O’Bryan et al., 1991; Faust et al., 1992; Jia andAlthough Gas 6 has originally been identified as a growth

arrest-specific gene (Schneider et al., 1988; Manfioletti et Hanafusa, 1994), and the same motif was also found in neu-ral cell adhesion molecules including NCAM. In addition,al., 1993), the only biological activity of Gas 6 which has

been reported thus far is growth potentiation of vascular the Ark receptor functions through homophilic binding,which leads to an increase in receptor phosphorylation (Bel-smooth muscle cells (Nakano et al., 1995) and Schwann

cells (Chen et al., 1996). Gas 6 is structurally related to losta et al., 1995). In the case of Ark, heterophilic interac-tion between Ark and unidentified surface molecules thatprotein S, a negative coregulator in the blood coagulation




FIG. 5. (A) Haplotype analysis of the interspecific backcross progeny carrying recombination breakpoint between D2Mit11 and D2Nds3.Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6 1 Mus spretus) F1parent. Black squares, the presence of both M. spretus and C57BL/6 alleles; hatched squares, the presence of either C57BL/6 or M. spretusalleles. Locus order was determined by minimizing the number of observed recombination breakpoints. The number of offspring inheritingeach type of chromosome is listed at the bottom of each column. (B) Linkage map of the proximal region of mouse chromosome 2constructed based on the analysis of the interspecific backcross (left) or on the Mouse Genome Database (Mouse Genome InformaticsProject, The Jackson Laboratory, Bar Harbor, Maine. World Wide Web URL: http://www.informatics.jax.org. January, 1995) (right). Geneticdistances are given in centimorgans (cM) and are shown for each pair of loci (left) or from centromere (right) on the left of the eachchromosome map. The positions of the underlined loci in human chromosomes are shown to the right of the chromosome maps. Referencesfor the human map positions of loci cited in this study can be obtained from the Genome Data Base, a computerized database of humanlinkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore, MD).

could have the same extracellular motif has also been sug- that cause depletion of spermatogonia. In vitro, removal ofgerm cells from cocultures of Sertoli cell and germ cells isgested (Bellosta et al., 1995). Based on the analogy of the

interaction of Ark, Sky may be involved in an interaction followed by a decrease in these proteins in Sertoli cells. More-over, addition of pachytene spermatocytes and early sperma-with identical or related molecules on the surface of adja-

cent cells, although the homophilic interaction of Sky has tids or medium conditioned with these cells to purified Ser-toli cells stimulates secretion of these proteins in culture.not been determined. The expression of Sky in PGCs and

adjacent pre-Sertoli cells in 12.5 and 14.5 dpc male genital From this evidence, soluble factors produced by germ cellsplay a key role in Sertoli cell function. In the case of Sky,ridges (Figs. 1A–1C) is compatible with the idea that Sky

mediates direct cell–cell interaction among these cells. In expression in Sertoli cells is markedly upregulated in germcell-deficient W/Wv mice compared with normal mice (Figs.addition, Sky expression was detected in Sl/Sl4 and Sl/Sl4-

m220 cells (data not shown), and this raises a possibility 1 and 4). Higher expression was also found in jsd/jsd testis(Fig. 4), in which only type A spermatogonia and Sertoli cellsthat Sky mediates direct interaction between PGCs and the

feeder cells to support PGC growth or survival in culture. exist in tubules, and in juvenile testes, in which spermato-cytes have not appeared. These observations indicate thatIn postnatal testis, germ cells, particularly pachytene

spermatocytes and early spermatids, modulate Sertoli cell spermatocytes and/or spermatid suppress Sky expression inSertoli cells. Feedback signals that repress some functions offunction (Jegou, 1993). Production of various proteins in Ser-

toli cells is induced by germ cells. For example, secretion of fully differentiated Sertoli cells may be provided by differ-entiating testicular germ cells (Yomogida et al., 1994).androgen binding protein, transferrin, and inhibin from Ser-

toli cells was decreased after treatment of anti-mitotic agents Sky expression was specifically detected in male genital




Bitgood, M. J., Shen, L., and McMahon, A. P. (1996). Sertoli cellridges after 11.5 dpc (Fig. 1 and data not shown), and thissignaling by Desert hedgehog regulates the male germline. Curr.suggests that the gene product is involved in male sexualBiol. 6, 298–304.differentiation, including differentiation of Sertoli cells.

Brannan, C. I., Lyman, S. D., Williams, D. E., Eisenman, J., Ander-The expression of Sky in only male PGCs also could haveson, D. M., Cosman, D., Bedell, M. A., Jenkins, N. A., and Cope-sex-specific consequences. For example, only male PGCsland, N. G. (1991). Steel-Dickie mutation encodes a c-Kit ligand

can give rise to teratomas and teratocarcinomas under par- lacking transmembrane and cytoplasmic domains. Proc. Natl.ticular conditions (Stevens, 1983), and the differential Acad. Sci. USA 88, 4671–4674.growth potential of male and female PGCs might be caus- Chen, R. L. J., Hammonds, G., Philips, H., Armanini, M., Wood, P.,ally related to Sky expression. Gas 6 expression was de- Bunge, R., Godowski, P. J., Sliwkowski, M. X., and Mather, J. P.tected not only in male but also in female genital ridges (1996). Identification of Gas 6 as a growth factor for human

Schwan cells. J. Neurosci. 16, 2012–2019.(Fig. 2). Although Sky expression was not detected in femaleCheng, L., Gearing, D. P., White, L. S., Compton, D. L., Schooley,genital ridges, we cannot exclude the possibility of low-

K., and Donovan, P. J. (1994). Role of leukemia inhibitory factorlevel expression which can not be detected by in situ hybrid-and its receptor in mouse primordial germ cell growth. Develop-ization. Alternatively, other Sky-related receptors to whichment 120, 3145–3153.Gas 6 can bind might be expressed in female genital ridges.

Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNAIn ovaries, a localized signal for Sky transcripts was found isolation by acid guanidium thiocyanate–phenol–chloroform ex-

in interstitial cells and cuboidal granulosa cells only at pre- traction. Anal. Biochem. 162, 156–159.natal and neonatal stages, which suggests involvement of Crosier, P. S., Lewis, P. M., Hall, L. R., Vitas, M. R., Morris, C. M.,Sky in ovarian development at very specific stages. Al- Beier, D. R., Wood, C. R., and Crosier, K. E. (1994). Isolation of athough the expression of Sky in adult ovaries was detected receptor tyrosine kinase (DTK) from embryonic stem cells: Struc-

ture, genetic mapping and analysis of expression. Growth Factorsby Northern blot, no specific signal was found using in situ11, 125–136.hybridization (data not shown), and it seems to be expressed

Dai, W., Pan, H., Hassanain, H., Gupta, S. L., and Murphy, M. J.weakly in the entire ovary.(1994). Molecular cloning of a novel receptor tyrosine kinase, tif,In this study, we mapped murine Sky to chromosome 2highly expressed in human ovary and testis. Oncogene 9, 975–between D2Mit17 and D2Mit164 and located it near979.D2Mit190 within the region of linkage homology with hu-

Davis, A. M. (1994). Tracking neurotrophin function. Nature 368,man chromosome 15 (Fig. 5B). Crosier et al. (1994) mapped193–194.

Sky (DTK) to band F on chromosome 2 by in situ hybridiza- De Felici, M., and Dolci, S. (1991). Leukemia inhibitory factor sus-tion, and they also mapped it near the b2-microglobulin tains the survival of mouse primordial germ cells cultured ongene by SSCP analysis in the BXD recombinant inbred (RI) TM4 feeder layers. Dev. Biol. 147, 281–284.series. Their results correlate well with our data presented De Felici, M., Dolci, S., and Pesce, M. (1993). Proliferation of mousehere which more precisely define the chromosomal position primordial germ cells in vitro: A key role for cAMP. Dev. Biol.

157, 277–280.of Sky. According to the mouse Genome Database and 1994Deng, C.-X., Wynshaw-Boris, A., Shen, M. M., Daugherty, C., Or-Mouse Chromosome Committee Reports, several uncloned

nitz, D. M., and Leder, P. (1994). Murine FGFR-1 is required formouse mutants are located around Sky, including Tsk, andearly postimplantation growth and axial organization. GenesIr2 (Fig. 5B). However, none of these mutations has a pheno-Dev. 8, 3045–3057.type which correlates with the expression pattern of Sky

Dietrich, W. F., Miller, J. C., Steen, R. G., Merchant, M., Damron,that we present in this paper.D., Nahf, R., Gross, A., Joyce, D. C., Wessel, M., Dredge, R. D.,Our results shown here indicate that Gas 6–Sky signalingMarquis, A., Stein, L. D., Goodman, N., Page, D. C., and Lander,

is important for PGC growth or survival and suggest that E. S. (1994). A genetic map of the mouse with 4,006 simple se-it is involved in development of gonads. Further studies are quence length polymorphisms. Nat. Genet. 7, 220–245.in progress to elucidate involvement of these molecules in Dolci, S., Williams, D. E., Ernst, M. K., Resnick, J. L., Brannan, C. I.,sexual differentiation and Sertoli cell function. Lock, L. F., Lyman, S. D., Boswell, H. S., and Donovan, P. J.

(1991). Requirement for mast cell growth factor for primordialgerm cell survival in culture. Nature 352, 809–811.ACKNOWLEDGMENTS

Faust, M., Ebensperger, C., Schulz, A. S., Schleithoff, L., Hameister,We thank Dr. Brigid Hogan for critical reading and comments H., Bartram, C. R., and Janssen, J. W. G. (1992). The murine ufo

and Dr. Kensaku Mizuno for helpful discussion. We also thank Dr. receptor: Molecular cloning, chromosomal localization and inMichael Rhodes for his help using EUCIB. This work was supported situ expression analysis. Oncogene 7, 1287–1293.by the grants-in-aid from the Ministry of Education, Science, and Feldman, B., Poueymirou, W., Papaioannou, V. E., DeChiara, T. M.,Culture of Japan and a research grant from the Princess Takamatsu and Goldfarb, M. (1995). Requirement of FGF-4 for postimplanta-Cancer Research Fund. tion mouse development. Science 267, 246–249.

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and spermatogenic cycle-specific expression of transcription fac- Accepted September 24, 1996



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