A Receptor Tyrosine Kinase, Sky, and Its Ligand Gas 6 Are Expressed In

DEVELOPMENTAL BIOLOGY 180, 499–510 (1996) ARTICLE NO. 0323 A Receptor Tyrosine Kinase, Sky, and View metadata, citation and similar papers at core.ac.uk brought to you by CORE

Its Ligand Gas 6 Are Expressed in Gonads and provided by Elsevier - Publisher Connector Support Primordial Germ Cell Growth or Survival 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 Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Osaka 565, Japan; ‡Department of Molecular Information, Institute of Molecular and Cellular Biosciences, Tokyo University, 1-1 Yayoi, Tokyo 113, Japan; and §Shionogi Research Laboratories, Shionogi & Co., Ltd., Osaka 553, Japan

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

INTRODUCTION matogonia (Yoshinaga et al., 1991; Rossi et al., 1993; Tajima et al., 1994) and for the maintenance of growing oocytes The proliferation and differentiation of germ cells are reg- (Packer et al., 1994). Other growth factors, including leuke- ulated by a number of environmental and intrinsic factors. mia inhibitory factor (LIF) (De Felici and Dolci, 1991; Mat- One of the most important is Steel factor, which is neces- sui et al., 1991), basic ﬁbroblast growth factor (bFGF), (Mat- sary not only for the proliferation and survival of primordial sui et al., 1992; Resnick et al., 1992), and tumor necrosis germ cells (PGCs) (Godin et al., 1991; Dolci et al., 1991; factor a, (TNFa), (Kawase et al., 1994), synergistically pro- Matsui et al., 1991), but also for the development of sper- mote PGC growth with each other in vitro. Activin A and IL-1a are also known to stimulate the growth of spermatogonia (Jegou, 1993). It is, however, not clear whether these 1 To whom correspondence should be addressed: Fax: 81-22-717- factors affect germ cells in vivo. Although PGCs express 8488. E-mail: [email protected]. LIF receptor (LIFR) on their surface (Cheng et al., 1994),

0012-1606/96 $18.00 Copyright ᭧ 1996 by Academic Press, Inc. All rights of reproduction in any form reserved. 499

AID DB 8417 / 6x16$$$601 11-20-96 23:33:10 dba AP: Dev Bio 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 to affinity LIFR gene (Ware et al., 1995) are fertile. OSM, a isolate such genes from a PGC-derived embryonic germ (EG) factor that shares the LIFR/gp130 complex as a specific re- cell line. One of these genes was specifically expressed in ceptor, also stimulates PGC growth in vitro (Cheng et al., gonads and brain in the adult mouse and was identical to 1994) and may be physiologically active in vivo. More re- Sky previously cloned from a hepatoma cell line, brain, and cently the physiological importance of soluble factors Des- a teratocarcinoma cell line (Ohashi et al., 1994; Mark et al., ret hedgehog (Bitgood et al., 1996) and BMP8b (Zhao et al., 1994; Lai et al., 1994; Crosier et al., 1994). Sky encodes a 1996), which are secreted from Sertoli cells and spermato- receptor tyrosine kinase, which has immunoglobulin-like cytes, respectively, has been reported. Null mutants for and fibronectin type III-like repeats in its extracellular do- these genes exhibit male germ cell deficiency indicating main that are characteristic of neural cell adhesion mole- that they are positive regulators for male germ cell growth, cules. It has recently been reported that Gas 6, originally survival, or differentiation in vivo. cloned from serum-starved NIH3T3 cells (Schneider et al., Direct interaction between germ cells and their support- 1988; Manfioletti et al., 1993), was a ligand for Sky. The ing cells is necessary for normal gonad development. Steel structural feature as a cell adhesion molecule and the spe- factor may be synthesized as either a soluble or membrane- cific binding with the ligand suggest that Sky could function associated form. The importance of the latter in vivo has through homophilic interaction as well as binding with Gas been shown by analysis of homozygous Sld mutant mice 6 (Godowski et al., 1995; Ohashi et al., 1995). We report which produce only the soluble form due to a structural here that Sky and Gas 6 are expressed in developing gonads alteration of the gene. Development of germ cells is im- and that recombinant Gas 6 can support PGC growth or paired in these mice (Flanagan et al., 1991; Brannan et al., survival in culture. 1991). A role of supporting cells for development of PGCs is also suggested by mice homozygous for a disruption of the Ftz-F1 gene, which encodes an orphan nuclear receptor. MATERIALS AND METHODS Ftz-F1 is expressed in pre-Sertoli cells and interstitial cells in 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 degeneration of the developing gonads. PGCs normally col- an reverse transcription-polymerase chain reaction-based strategy onize the developing genital ridges by 11.5 dpc in the mu- as described (Matsubara et al., 1995). To obtain longer cDNAs, a tant, but markedly diminished numbers of PGCs are found murine adult Balb/c brain cDNA library (Stratagene) was screened at later stages. Although precise functions of Ftz-F1 in de- using the PCR clone as a probe, and 20 overlapping clones were veloping genital ridges are not well defined, the results sug- obtained. The longest had a 3.3-kb insert and contained most of the coding region and 3؅ untranslated region. The 5؅ region of Sky gest that the failure of PGC growth is caused by improper cDNA 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 sequences For 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 is therefore of interest to identify molecules involved in (DIG)-labeled RNA probe was carried out essentially as described germ cell-supporting cell interaction. (Hirota et al., 1992). All embryos and gonads except WBB6F1-W/ The importance of receptor tyrosine kinases (RTKs) and Wv testis were obtained from Balb/c mice and were dissected ac- their ligands in cell–cell communication during develop- cording to Hogan et al. (1995). Tissues were fixed in 4% paraformal- ment has been amply demonstrated. For example, c-Kit and dehyde and embedded in paraffin. Five-micrometer sections were dewaxed, rehydrated, and pretreated with 20 mg/ml proteinase K its 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 sections as well as germ cells (Williams et al., 1992). Other examples were then hybridized at 50ЊC for 16 h in 50% formamide, 10 mM are 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 embryo (Feldman et al., 1995; Yamaguchi et al., 1994; Deng slides were then washed at 65ЊC for 30 min in 50% formamide, et al., 1994), and platelet-derived growth factor (PDGF) re- 21 SSC, and treated with 10 mg/ml RNaseA at 37ЊC for 30 min, ceptor a, which plays a critical role in the differentiation followed by washes in 21 SSC and 0.21 SSC at 50ЊC. After washing, of mesodermal and neural crest-derived mesenchyme (Ste- slides were incubated with anti-DIG–alkaline phosphatase conju- phenson et al., 1991; Schatteman et al., 1992). Members of gate for 30 min and then reacted with 4-nitroblue tetrazolium chlo- ride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate, 4-toluidine the Trk receptor family and their ligand neurotrophins are salt (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, rabbit ing development. anti-rat Gas 6 polyclonal antibody which was raised against the To identify receptor tyrosine kinases that are expressed purified rat Gas 6 was used. Cryosections of tissues from Balb/c

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mice and embryos were fixed in PLP fixative and were treated in turing polyacrylamide gel and photographed after staining with peroxoblock (Zymed). Sections were stained using a Histostain SP ethidium bromide. Kit (Zymed) according to the instruction of the manufacturer and were counterstained with hematoxylin. The concentration of the primary antibody was 5 mg/ml. Primary culture of PGCs. Primary culture of murine PGCs was RESULTS carried out as described (Matsui et al., 1991, 1992). The caudal region of 8.5 dpc embryos obtained from ICR1 B6D2F1 mating was Cloning and Structural Analysis of GCK4 (Sky) dissociated into single cells by trypsin treatment. Cells from the cDNA equivalent of 0.5 embryos were seeded into a well containing Sl/ Sl4-m220 cells or Sl/Sl4 cells as feeder layers (Matsui et al., 1991, To isolate cDNAs encoding receptor tyrosine kinases that 1992). Sl/Sl4 is a stromal cell line derived from the hematopoietic play a role in germ cell development, we employed a PCR- microenvironment of a homozygous Sl/Sl murine embryo in which based method using RNA obtained from EG cells and prim- the entire coding sequence of Steel factor gene is deleted (Toksoz ers corresponding to a conserved region in the kinase do- et al., 1992). Sl/Sl4-m220 is a derivative of Sl/Sl4, which expresses main as described (Matsubara et al., 1995). One of the clones only the alternatively spliced form of mouse Steel factor in which (GCK4) was found to be predominantly expressed in the the extracellular proteolytic cleavage site encoded by exon 6 is testis, ovary, and brain in the adult mouse by Northern blot missing (Matsui et al., 1991). Rat recombinant Gas 6 was added at hybridization (data not shown). We next screened a brain a concentration of 350 ng/ml, which was shown to be optimal for cDNA library with the PCR fragment of GCK4, and the PGC growth. .longest 3.3-kb cDNA and 5؅-RACE clones were sequenced Northern blot hybridization. Total RNAs were prepared from v Consequently, we found that GCK4 was identical with pre- testes of postnatal ICR mice, 3- to 4-month-old WBB6F1-W/W mice, and 4.5-month-old B6-jsd/jsd mice by acid guanidium thiocy- viously isolated cDNAs designated human Sky (Ohashi et anate–phenol–chloroform (AGPC) method (Chomczynski and Sac- al., 1994), human and mouse rse (Mark et al., 1994), mouse chi, 1987). The germ cell-rich fraction and Leydig cell-rich and Tyro3 (Lai et al., 1994), and human and mouse DTK (Croiser 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 has tion 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 blot hybridization was carried out under high stringency conditions ac- 32 cording to Sambrook et al. (1989). A P-labeled 967-bp SacI frag- Sky Is Differentially Expressed in Male and Female ment of Sky cDNA was used for a probe. Embryonic and Neonatal Gonads Interspecific backcross mapping. The large backcross panel between the two mouse species Mus spretus and We first examined the distribution of Sky transcripts in C57BL/6 containing 1000 animals was generated jointly by embryonic and neonatal gonads by in situ hybridization. At the Human Genome Project Resource Center, UK, and the 10.5 dpc, PGCs migrate through the dorsal mesentery to- Pasteur Institut, France. (C57BL/6 1 M. spretus) F1 females ward the developing genital ridges. Some cells along the were backcrossed to C57BL/6 or M. spretus males. Each dorsal mesentery and around the dorsal aorta showed weak backcross progeny mouse has been scored for three to four signal (data not shown). At 11.5 dpc, no significant signal microsatellite markers per chromosome, completing an an- above background was observed in both male and female chor map of 70 loci across the mouse genome. From 873 genital ridges (data not shown). backcross progeny, a total 62 animals, which are recombi- Abundant expression of Sky transcripts was seen in 12.5 nants between D2Mit11 and D2Nds3 on chromosome 2, and 14.5 dpc male genital ridges (Figs. 1A–1C). The signal were used to determine the precise chromosomal location was distributed in both PGCs, identified by their relatively of Sky. large and round nuclei, and adjacent pre-Sertoli cells. No Southern blot hybridization and PCR amplification. DNA (5 signal was found outside of the testicular cords where inter- 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 to prepared 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 94ЊC for 4 min followed by 30 recognizable by their round nuclei. They were positive for cycles of 94ЊC for 1 min, 55ЊC for 1 min, 72ЊC for 1 min, and a final elongation step at 72ЊC for 7 min. Taq polymerase (Amersham) was Sky expression, although the signal was weak. Stronger sig- used under assay conditions specified by the manufacturer. The nal was seen around the nuclei of Sertoli cells that are pe- concentration of each primer was 0.7 mM. Microsatellite marker ripherally located in the testis cords (Fig. 1D). At birth, Sky primer sets (Map Pairs) were purchased from Research Genetics transcripts were detected in the center of the cord that (Huntsville, AL). PCR products were separated on a 8% nondena- mainly contains Sertoli cell cytoplasm. Gonocytes that

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11-20-96 23:33:10 dba AP: Dev Bio 504 Matsubara et al. have large and round nuclei in the cord were negative (data 1995). In contrast, Gas 6 showed no effect on PGCs cultured not shown). on the feeder layer of Steel factor-lacking Sl/Sl4 cells 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 in birth, cuboidal cells surrounding follicles that had initiated growth were positive in the central part of the ovaries (Fig. Postnatal Testes, While Differentiated Testicular Germ Cells Suppress Its Expression 1I), 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- 1 week of age, no localized signal was found in the ovaries riphery of the testis cords, and the central region where (data not shown). Sky expression was prominent only contains Sertoli cell cytoplasm (data not shown). Little or no expression was seen 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 restricted Supports PGC Growth or Survival in Culture around the basally located nuclei of Sertoli cells in all semi- We next examined the expression of Gas 6, a ligand for niferous tubules, and signal was markedly diminished (data Sky, in murine gonads by immunohistochemistry with not shown). The same pattern of expression was maintained anti-Gas 6 antibody. At 11.5 dpc no significant signal above from 35 days of age onward (Fig. 1E). To determine the time background was observed in both male and female genital course of expression of Sky transcripts in developing testes, ridges (Figs. 2A and 2G). Strong staining was seen in intersti- we prepared RNA from postnatal testes and carried out tial cells which were surrounding Sky-expressing cells in Northern blot hybridization. In agreement with the results male genital ridges at 12.5 and 14.5 dpc (Figs. 2B–2D). of in situ hybridization, the level of Sky transcripts gradu- Weaker signal was also detected in female genial ridges ally diminished after 10 days of age (Fig. 4). Sertoli cell- (Figs. 2H and 2I). In adults, Gas 6 existed in peritubular cells specific expression of Sky in adult testis was confirmed by and endothelial cells in testes (Figs. 2E and 2F) and in theca Northern blot hybridization using RNA prepared from frac- cells and corpus luteum in ovaries (data not shown). tionated testicular cells. Significant expression was seen in The expression of both the receptor and the ligand in the tubule fraction that contains Sertoli cells exclusively, genital ridges suggests that they are involved in PGC growth while only faint signal was found in germ cell and Leydig or survival. To investigate this, we tested the effect of re- cell fractions (Fig. 4). A higher level of Sky expression was combinant Gas 6 on PGC growth in culture. We used PGCs detected in the germ cell-deficient testis of W/Wv mice (Fig. obtained from 8.5 dpc embryos, which show similar respon- 4). This was not due to the difference in Sertoli cell number sibility to various growth factors as gonadal PGCs (Matsui between the mutant and normal mice, because expression et al., 1991, 1992; Godin et al., 1991; De Felici and Dolci, in each Sertoli cell in the mutant testis was stronger than 1991; De Felici et al., 1993). PGCs increased in number on that detected in normal testis by in situ hybridization (Fig. the feeder layer of Sl/Sl4-m220 cells expressing membrane- 1F). Upregulated expression of Sky in testes was also ob- associated Steel factor without addition of Gas 6, but the served in jsd/jsd (juvenile spermatogonial depletion) mice number of PGCs was significantly higher in the presence by Northern blot (Fig. 4). In jsd/jsd mice, the first wave of of Gas 6 than that in controls at 4 days in culture (Fig. 3A), spermatogenesis occurs, but only type A spermatogonia and and the increase was dose-dependent with an optimum at Sertoli cells are found in the seminiferous tubules after the 350 ng/ml (Fig. 3C). The optimum concentration was simi- juvenile stage due to the inability of type A spermatogonia 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 14.5 dpc (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 14.5 dpc (G), and ovaries at 17.5 dpc (H) and 2 days after birth (I) were hybridized with DIG-labeled antisense RNA probe specific for mouse Sky. Sky was expressed in the cells in forming testicular cords (t), whereas no significant signal above background was found in female genital ridges (A –C, G). Signal on mesonephros was nonspecific. Positive signal was also seen around presumptive gonocytes (gc) and Sertoli cells (s) in the testicular cords at 17.5 dpc (D). Peripherally located Sertoli cells gave weak signal at 35 days (E), whereas much stronger signal was found in Sertoli cells in W/Wv testis (F). In ovaries, weak but significant signal above background was seen in interstitial cells (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 stained with 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.

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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 ﬁxed and the number of alkaline phosphatase positive cells was counted on Day 1 and Day 4 in culture. Each experiment was carried out with duplicate wells, and numbers are the means { SEM of eight (A), six (B), and one (C) separate experiment. *Signiﬁcantly different from control 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 recombinants To investigate whether the Sky gene is localized to a between D2Mit11 and D2Nds3 from 873 backcross progeny. region of the mouse genome involved in gonad develop- These recombinants were further typed for D2Mit42, ment, fine chromosomal mapping was carried out using an D2Mit103, D2Mit17, D2Mit190, Sky, and D2Mit164. The interspecific backcross panel. genetic order of this region is determined by the haplotype C57BL/6 and M. spretus genomic DNAs were digested analysis shown in Fig. 5A. The genetic order and distances with several different restriction enzymes and analyzed by ({1 standard error) are as follows: Cen---D2Mit11-11.77 { Southern blot hybridization for restriction fragment length 1.90 cM-D2Mit42-4.84 { 1.26 cM-D2Mit103-0.35 { 0.35 polymorphisms (RFLPs). An informative RFLP was obtained cM-D2Mit17-0.35 { 0.35 cM-Sky/D2Mit190-0.69 { 0.49 following digestion with BglII, yielding a 6.4-kb hybridizing cM-D2Mit164-3.56 { 1.08 cM-D2Nsd3. A linkage map of band in M. spretus and a 4.4-kb band in C57BL/6 (data not the loci included in this study on chromosome 2 is shown in 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 phenotype spretus-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 of the linkage relationship of the Sky gene. This analysis DISCUSSION mapped Sky to mouse chromosome 2 between D2Mit11 and We have cloned a cDNA encoding Sky receptor tyrosine D2Nds3. To reveal the chromosomal location of Sky more kinase from EG cells and have found that the gene was

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FIG. 4. Detection of Sky mRNA by Northern blot hybridization in developing and germ cell-deﬁcient testes and fractionated testicular cells. 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 reprobed with 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; after 11.5 dpc (when Sertoli cell differentiation starts), and Ohashi et al., 1995). Our results (Figs. 1 and 2) suggest that in prenatal testes, but the expression was not detected in Gas 6 produced in interstitial cells in genital ridges and female genital ridges by in situ hybridization (Fig. 1). We adult testes diffuses in the extracellular space to interact have also found that Gas 6, a ligand for Sky, was expressed with Sky expressed on PGC and Sertoli cell surfaces to sup- in both male and female genital ridges after 11.5 dpc (Fig. port their growth and/or functions. Although the recombi- 2) and that the recombinant Gas 6 supported PGC growth nant Gas 6 supported PGC growth or survival on mem- or survival in primary culture (Fig. 3). At later stages, Sky brane-bound Steel factor-expressing Sl/Sl4-m220 cells in is expressed only in supporting cells, i.e., Sertoli cells in culture (Fig. 3A), it had no effect on PGCs cultured on Sl/ testes and granulosa cells in ovaries (Figs. 1 and 4), and Gas Sl4 cells which lacked the Steel factor gene (Fig. 3B). This 6 was expressed in peritubular cells in testis (Fig. 2). We indicates that Gas 6 enhances Steel factor-induced growth concluded that the expression of Sky in Sertoli cells was or survival of PGCs. A similar effect of Gas 6 has been found suppressed by differentiated testicular germ cells for the on vascular smooth muscle cells, but in this case, Gas 6 following reasons: (1) The expression in developing testes enhances proliferation of the cells, which is induced by was diminished after 10 days of age, when spermatogenesis growth factors activating Ca2/-mobilizing receptors (Na- begins (Figs. 1 and 4). (2) Sky expression in Sertoli cells was kano et al., 1995). markedly upregulated in testes of germ cell-deficient W/Wv From the structural feature of Sky, there is another possi- and jsd/jsd mice whose spermatogonia fail to develop (Figs. ble model for Sky function. The extracellular domain of Sky 1 and 4). Taken together, our findings suggest that Gas 6– is composed of two immunogloblin-like domains and two Sky signaling contributes to gonad development in many fibronectin type III domains that are similar to the extracel- aspects. lular domains of receptor tyrosine kinase, Axl/UFO/Ark Although Gas 6 has originally been identified as a growth and Eyk (O’Bryan et al., 1991; Faust et al., 1992; Jia and arrest-specific gene (Schneider et al., 1988; Manfioletti et Hanafusa, 1994), and the same motif was also found in neu- al., 1993), the only biological activity of Gas 6 which has ral cell adhesion molecules including NCAM. In addition, been reported thus far is growth potentiation of vascular the Ark receptor functions through homophilic binding, smooth muscle cells (Nakano et al., 1995) and Schwann which leads to an increase in receptor phosphorylation (Bel- cells (Chen et al., 1996). Gas 6 is structurally related to losta et al., 1995). In the case of Ark, heterophilic interac- protein S, a negative coregulator in the blood coagulation tion between Ark and unidentified surface molecules that

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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)F1 parent. Black squares, the presence of both M. spretus and C57BL/6 alleles; hatched squares, the presence of either C57BL/6 or M. spretus alleles. Locus order was determined by minimizing the number of observed recombination breakpoints. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. (B) Linkage map of the proximal region of mouse chromosome 2 constructed based on the analysis of the interspecific backcross (left) or on the Mouse Genome Database (Mouse Genome Informatics Project, The Jackson Laboratory, Bar Harbor, Maine. World Wide Web URL: http://www.informatics.jax.org. January, 1995) (right). Genetic distances are given in centimorgans (cM) and are shown for each pair of loci (left) or from centromere (right) on the left of the each chromosome map. The positions of the underlined loci in human chromosomes are shown to the right of the chromosome maps. References for the human map positions of loci cited in this study can be obtained from the Genome Data Base, a computerized database of human linkage 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 of gested (Bellosta et al., 1995). Based on the analogy of the germ cells from cocultures of Sertoli cell and germ cells is interaction of Ark, Sky may be involved in an interaction followed by a decrease in these proteins in Sertoli cells. More- with identical or related molecules on the surface of adja- over, addition of pachytene spermatocytes and early sperma- cent cells, although the homophilic interaction of Sky has tids or medium conditioned with these cells to purified Ser- not been determined. The expression of Sky in PGCs and toli cells stimulates secretion of these proteins in culture. adjacent pre-Sertoli cells in 12.5 and 14.5 dpc male genital From this evidence, soluble factors produced by germ cells ridges (Figs. 1A–1C) is compatible with the idea that Sky play a key role in Sertoli cell function. In the case of Sky, mediates direct cell–cell interaction among these cells. In expression in Sertoli cells is markedly upregulated in germ addition, Sky expression was detected in Sl/Sl4 and Sl/Sl4- cell-deficient W/Wv mice compared with normal mice (Figs. m220 cells (data not shown), and this raises a possibility 1 and 4). Higher expression was also found in jsd/jsd testis that Sky mediates direct interaction between PGCs and the (Fig. 4), in which only type A spermatogonia and Sertoli cells feeder cells to support PGC growth or survival in culture. exist in tubules, and in juvenile testes, in which spermato- In postnatal testis, germ cells, particularly pachytene cytes have not appeared. These observations indicate that spermatocytes and early spermatids, modulate Sertoli cell spermatocytes and/or spermatid suppress Sky expression in function (Jegou, 1993). Production of various proteins in Ser- Sertoli cells. Feedback signals that repress some functions of toli cells is induced by germ cells. For example, secretion of fully differentiated Sertoli cells may be provided by differ- androgen binding protein, transferrin, and inhibin from Ser- entiating testicular germ cells (Yomogida et al., 1994). toli cells was decreased after treatment of anti-mitotic agents Sky expression was specifically detected in male genital

AID DB 8417 / 6x16$$$603 11-20-96 23:33:10 dba AP: Dev Bio 508 Matsubara et al. ridges after 11.5 dpc (Fig. 1 and data not shown), and this Bitgood, M. J., Shen, L., and McMahon, A. P. (1996). Sertoli cell suggests that the gene product is involved in male sexual signaling by Desert hedgehog regulates the male germline. Curr. differentiation, including differentiation of Sertoli cells. Biol. 6, 298–304. The expression of Sky in only male PGCs also could have Brannan, C. I., Lyman, S. D., Williams, D. E., Eisenman, J., Ander- son, D. M., Cosman, D., Bedell, M. A., Jenkins, N. A., and Cope- sex-specific consequences. For example, only male PGCs land, 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 (Fig. 2). Although Sky expression was not detected in female Schwan cells. J. Neurosci. 16, 2012–2019. genital ridges, we cannot exclude the possibility of low- Cheng, L., Gearing, D. P., White, L. S., Compton, D. L., Schooley, level expression which can not be detected by in situ hybrid- K., and Donovan, P. J. (1994). Role of leukemia inhibitory factor ization. Alternatively, other Sky-related receptors to which and its receptor in mouse primordial germ cell growth. Develop- Gas 6 can bind might be expressed in female genital ridges. ment 120, 3145–3153. Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA In 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 a though the expression of Sky in adult ovaries was detected receptor tyrosine kinase (DTK) from embryonic stem cells: Struc- by Northern blot, no specific signal was found using in situ ture, genetic mapping and analysis of expression. Growth Factors hybridization (data not shown), and it seems to be expressed 11, 125–136. weakly in the entire ovary. Dai, W., Pan, H., Hassanain, H., Gupta, S. L., and Murphy, M. J. In this study, we mapped murine Sky to chromosome 2 (1994). Molecular cloning of a novel receptor tyrosine kinase, tif, between D2Mit17 and D2Mit164 and located it near highly expressed in human ovary and testis. Oncogene 9, 975– D2Mit190 within the region of linkage homology with hu- 979. Davis, A. M. (1994). Tracking neurotrophin function. Nature 368, man chromosome 15 (Fig. 5B). Crosier et al. (1994) mapped 193–194. 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 on gene 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 mouse here which more precisely define the chromosomal position primordial germ cells in vitro: A key role for cAMP. Dev. Biol. of Sky. According to the mouse Genome Database and 1994 157, 277–280. Mouse Chromosome Committee Reports, several uncloned Deng, C.-X., Wynshaw-Boris, A., Shen, M. M., Daugherty, C., Or- mouse mutants are located around Sky, including Tsk, and nitz, D. M., and Leder, P. (1994). Murine FGFR-1 is required for Ir2 (Fig. 5B). However, none of these mutations has a pheno- early postimplantation growth and axial organization. Genes type which correlates with the expression pattern of Sky Dev. 8, 3045–3057. that we present in this paper. Dietrich, W. F., Miller, J. C., Steen, R. G., Merchant, M., Damron, D., Nahf, R., Gross, A., Joyce, D. C., Wessel, M., Dredge, R. D., Our results shown here indicate that Gas 6–Sky signaling Marquis, 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 primordial ACKNOWLEDGMENTS germ cell survival in culture. Nature 352, 809–811. 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 in Michael 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. 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