Developmental expression pattern of Stra6, a retinoic acid-responsive gene encoding a new type of membrane protein

Mechanisms of Development 63 (1997) 173–186

Developmental expression pattern of Stra6, a retinoic acid-responsive gene encoding a new type of membrane protein Philippe Bouillet, Vincent Sapin, Claire Chazaud, Nadia Messaddeq, Didier De´cimo, Pascal Dolle´, Pierre Chambon* Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, CNRS/INSERM/ULP, Colle`ge de France, B.P. 163, 67404 Illkirch Cedex, C.U. de Strasbourg, France Received 20 January 1997; revised version received 10 March 1997; accepted 11 March 1997

Abstract Retinoic acid plays important roles in development, growth and differentiation by regulating the expression of target genes. A new retinoic acid-inducible gene, Stra6, has been identified in P19 embryonal carcinoma cells using a subtractive hybridization cDNA cloning technique. Stra6 codes for a very hydrophobic membrane protein of a new type, which does not display similarities with previously characterized integral membrane proteins. Stra6, which exhibits a specific pattern of expression during development and in the adult, is strongly expressed at the level of blood-organ barriers. Interestingly, in testis Sertoli cells, Stra6 has a spermatogenic cycle-dependent expression which is lost in testes of RARa null mutants where Stra6 is expressed in all tubules. We suggest that the Stra6 protein may be a component of an as yet unidentified transport machinery.  1997 Elsevier Science Ireland Ltd. Keywords: Mouse development; Mesoderm; Nervous system; Retinoids; Testis; Transmembrane proteins

1. Introduction Retinoids, particularly retinoic acid (RA), play key roles in cellular proliferation and differentiation, as well as in development (Blomhoff, 1994; Linney and LaMantia, 1994; Sporn et al., 1994; Kastner et al., 1995; and references therein). RA exerts its effects through ligand-inducible transcription regulators belonging to the superfamily of nuclear receptors. Three types of retinoic acid receptors (RARa, b and g) bind and respond to all-trans (T-RA) and 9-cis (9C-RA) isomers of RA, whereas the retinoid X receptors (RXRa, b and g) can only bind and respond to 9CRA. RARs and RXRs modulate gene expression by binding as heterodimers to RA responsive elements located in the regulatory regions of target genes (reviewed in Chambon, 1994, 1996; Kastner et al., 1994, 1995; Mangelsdorf et al., 1994; Mangelsdorf and Evans, 1995).

* Corresponding author. Tel: +33 3 88653213/15/10; fax: +33 3 88653203; e-mail: [email protected]

0925-4773/97/$17.00  1997 Elsevier Science Ireland Ltd. All rights reserved P II S0925- 4773 (97 )0 0039- 7

During differentiation, RA induces a change in the cell morphology and the expression of a number of genes (Gudas et al., 1994). Using a subtractive hybridization technique, we have isolated several cDNAs corresponding to genes whose expression is increased during RA-induced differentiation of embryonal carcinoma (EC) P19 cells (Bouillet et al., 1995). We describe here the structure and expression features of one of these genes, Stra6, which encodes a novel type of integral transmembrane protein highly expressed during development and in blood-organ barriers.

2. Materials and methods 2.1. Cell culture and RA treatment Mouse embryonal carcinoma (EC) P19 cells were grown as monolayers in Dulbecco’s medium supplemented with 5% fetal calf serum and maintained as described (Rudnicki et al., 1988). F9 EC cells and D3 embryonic stem (ES) cells

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were also cultured as described (Lufkin et al., 1991; Wang et al., 1985). P19 and F9 cells were induced to differentiate by treatment for 24 h with 10−6 M T-RA, whereas 10−8 M was used for D3 ES cells.

facturer’s instructions. As controls, preimmune sera were used in place of the antibodies. All slides were slightly counterstained with methyl green. 2.5. In situ hybridization

2.2. RNA extraction and analysis Total RNA was isolated from cells or from adult mouse or bovine tissues using the LiCl/urea/acid phenol extraction method (Auffray and Rougeon, 1980), followed by removal of contaminating DNA by sodium acetate precipitation. RNA and DNA amounts were determined by OD measurement. For Northern blotting experiments, 20 mg of total cellular RNA were fractionated by electrophoresis on 1% formaldehyde/formamide gel and transferred to a nylon membrane. After hybridization, the filters were washed with 0.1 × SSC, 0.1% SDS for 15 min at 65°C. Reverse transcriptase (RT)-PCR assays were performed as described (Bouillet et al., 1995). 2.3. cDNA cloning and sequencing Stra6 cDNA was isolated by subtractive hybridization/ differential screening as described (Bouillet et al., 1995). Full length cDNA was obtained by screening an oligo(dT)-primed P19 cDNA library, using the original Stra6 partial cDNA as a probe. The DNA sequence was determined on both strands using dyedeoxy terminator cycle sequencing on an ABI373A automated DNA sequencer (Applied Biosystems, Foster City, CA) or manually with a T7 DNA polymerase based kit. A DNA homology search was performed at the National Center for Biotechnology Information using the FASTA and BLAST network services (Bethesda, MD).

The 2993 nucleotide-long Stra6 cDNA cloned in pBluescript SK(−) (Stratagene) was used as template for in vitro T7 RNA polymerase transcription reactions including digoxygenin-11-UTP (Boehringer) or [35S]-CTP (Amersham), to produce antisense riboprobes. Probe synthesis, in situ hybridization (ISH) to sections of frozen embryos and placentas, and whole-mount ISH were performed as described (De´cimo et al., 1995). 2.6. Electron microscopy Adult male CD1 mice were anesthetized and perfused intra-aortically with a fixative consisting of 4% paraformaldehyde, 0.2% picric acid and 0.1% glutaraldehyde in phosphate-buffered saline. Testes were removed and kept overnight at 4°C in the same fixative, then immersed for an additional 20 h at 4°C in PBS. Stra6 immunodetection was performed on 100 mm sections by using the Vectastain ABC-Elite and DAB kits. Stained sections were fixed with 5% glutaraldehyde in PBS, postfixed in 1% osmium tetroxide for 1 h and dehydrated with ethanol and propylene oxide. After overnight infiltration in Epon resin, sections were flattened between microscope slides, polymerized at 60°C for 24 h, and finally glued onto plastic blocks. Sections (70 nm) were cut and collected on 200 mesh uncoated grids and examined with a Philips 208 electron microscope at 60 kV without counterstaining.

2.4. Antibodies and immunohistochemical procedures

3. Results

To raise antibodies against the Stra6 protein, a cDNA fragment encompassing amino acid positions 562–670 was inserted into the BamHI site of the pET15b bacterial expression vector (Novagen). The 6xHis-tagged recombinant protein was expressed in E. coli BL21, purified by affinity chromatography on Ni2+-NTA columns (Qiagen) and used to immunize rabbits. IgGs were precipitated with ammonium sulfate and further purified by affinity chromatography on two synthetic peptides corresponding to amino acids 586–615 and 620–647 of the Stra6 protein. The antibody against Stra8 has been described elsewhere (OuladAbdelghani et al., 1996). Stra6 and Stra8 were immunolocalized according to the ABC immunoperoxydase method using the Vectastain ABC-Elite and DAB kits (Vector Laboratories, Burlingame, CA). Tissue sections (10 mm) were fixed with cold acetone for 5 min (for Stra6 detection) or with cold acetone for 5 min followed by 4% formaldehyde for 15 min at 4°C (for Stra8 immunolocalization) and processed according to the manu-

3.1. Stra6 cDNA and genomic organization Using a differential subtractive hybridization strategy, we have identified 50 different cDNA fragments corresponding to RNAs whose levels are increased by T-RA treatment in P19 EC cells (Bouillet et al., 1995). One of these cDNA fragments (Stra6, 263 nucleotides) was used to screen an oligo(dT)-primed cDNA library prepared from T-RA-treated P19 cells, yielding three additional clones, the longer being 2993 nucleotides in length (Fig. 1A). This cDNA contains a single open reading frame corresponding to a 670 amino acid protein (74 kDa), starting with a methionine initiator codon at position 350 and terminated by a TGA stop codon at nucleotide 2360 (Fig. 1A). The sequence located 5′ of the initiation site contains one in-frame stop codon at position 302 and a putative polyadenylation signal (AATAAA) is present in the 3′ untranslated region (Fig. 1A, underlined). Hydropathy analysis of the predicted amino acid sequence reveals that it displays a high content of

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Fig. 1. (A) Nucleotide and deduced amino acid sequence of mouse Stra6 cDNA. Numbers on the left and right sides refer to the positions of nucleotides and amino acid residues, respectively. The upstream stop codon and the putative polyadenylation signal are underlined. Arrowheads indicate the position of exon boundaries. (B) Hydrophobicity plot of the deduced amino-acid sequence according to Kyte and Doolittle. Note the high content of hydrophobic residues.

hydrophobic residues (.50%) and contains nine potential transmembrane domains (a.a. 49–70, 98–118, 148–186, 206–223, 301–320, 364–383, 440–460, 475–493 and 514–539; Fig. 1B). Searches through the EMBL, Genbank and Swissprot databases with the FASTA and BLAST algorithms failed to reveal any closely related sequences. High stringency hybridization of a Stra6 cDNA probe on total RNA from mouse genital tract or bovine retina revealed a single RNA species of ~3 kb in size (Fig. 2A), indicating that our longest cDNA is nearly full length and that the Stra6 gene is conserved in other mammals. In vitro transcription/translation experiments using a reticulocyte lysate system (TNT kit, Promega) and the longest cDNA sequence generated a protein with an apparent molecular weight of 74 ± 1 kDa (Fig. 2B), consistent with that deduced from the cDNA sequence. To determine the genomic organization of the Stra6 gene, a mouse genomic library was screened using the 3 kb Stra6 cDNA as a probe. Several overlapping clones were isolated and shown to contain, over 30 kb, all the exons correspond-

ing to the sequence of our longest cDNA. There are 19 exons of variable size, the shortest being 24 and the longest 801 nt long. The positions of exon junctions are indicated in Fig. 1A (arrowheads). All the intron-exon boundaries matched consensus splice sites (data not shown). The 1.4 kb genomic sequence located upstream to exon 1 was determined (data not shown) and did not display features of eukaryotic gene promoters. As a putative splice acceptor sequence was present in the genomic DNA 11 bp upstream of the position corresponding to the 5′ end of the cDNA, we performed primer extension assays to determine whether the transcription initiation site corresponds to the 5′ end of the cDNA. No products longer than that expected from our longest cDNA were obtained. Therefore, a 4.5 kb genomic DNA fragment located immediately upstream of exon 1 was cloned in front of the chloramphenicol acetyl transferase (CAT) cDNA in the pBLCAT6 reporter plasmid (Boshart et al., 1992). This construct was transfected in several cell lines, in the absence or presence of RAR and RXR expression plasmids and their cognate ligands. No promoter activ-

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which prevent passive diffusion of many biomolecules from the blood. These barriers are made of epithelial or endothelial cells closely bound together by tight junctions and control the facilitative transport processes for monosaccharides, amino acids, fatty acids, vitamins and hormones from the blood stream to these tissues. Interestingly, the Stra6 gene was found to be expressed in these blood-organ barriers.

Fig. 2. (A) Northern blot detection of Stra6 RNA. Total RNAs were extracted from adult mouse female genital tract (1), bovine retinal pigment epithelium (2) and bovine neural retina (3). Twenty micrograms each of these RNAs were analyzed by Northern blotting using [32P]-labeled Stra6 cDNA as a probe. The positions of 28S and 18S rRNAs are indicated. The amount of RNA loaded in each lane was normalized with respect to 36B4 RNA which is similarly expressed in all cells and tissues (Bouillet et al., 1995; data not shown). Staining of the gel before Northern blotting also indicated that similar amounts of 18S and 28S RNA were present in all lanes. (B) SDS-PAGE analysis of in vitro transcription/translation products from full-length Stra6 cDNA. In vitro transcription and translation of the Stra6 cDNA was performed according to the instructions of the supplier of the reticulocyte lysate system (TNT kit, Promega).

ity could be detected (data not shown). These negative results indicate that essential enhancer sequences may be missing in this construct, or alternatively, that our primer extension assays failed to identify additional 5′-UTR sequence(s) located upstream of the 4.5 kb genomic DNA fragment. 3.2. Stra6 RNA expression Reverse transcriptase-coupled PCR (RT-PCR) was used to estimate the accumulation of Stra6 RNA upon RA treatment in several cell lines. In P19 EC cells, Stra6 transcripts accumulated in a time- and RA concentration-dependent manner (Fig. 3A). An increase in Stra6 RNA level was first detected 2 h after treatment of P19 cells with either 10 nM or 1 mM T-RA and reached a plateau level at 12– 24 h in cells treated with 1 mM T-RA (Fig. 3A). Expression of the Stra6 gene was also enhanced by T-RA treatment in D3 ES and F9 EC cells (Fig. 3B). In the adult mouse, expression of the Stra6 gene was detected at relatively high levels in several tissues such as brain, kidney, spleen, testis and female genital tract, whereas it was much lower or undetectable in heart, lung and liver (Fig. 3C). Polyclonal antibodies as well as in situ hybridization with antisense riboprobes were used to further define the sites of Stra6 expression in these various organs. 3.3. Stra6 expression in blood-organ barriers Several organs such as brain, eye and testis are kept in a specialized environment by means of selective membranes

Fig. 3. (A) Time course of Stra6 transcript accumulation in P19 cells incubated with ethanol (W), 10 nM T-RA (O) or 1 mM T-RA (A). The autoradiograms were quantitated with a phosphoimager system; RNA levels were normalized to the content of the invariant 36B4 ribosomal phosphoprotein RNA and expressed as a percentage of the maximal value. (B) Analysis of Stra6 expression in cultured cells. D3 ES cells were grown in the presence of 10 nM T-RA for 12, 24 or 48 h; 0 refers to cells grown with ethanol for 24 h. F9 cells were grown in the presence of ethanol (0) or 1 mM T-RA for 24 h. (C) Stra6 RNA expression in mouse adult organs. Relative Stra6 transcript levels were determined in brain (Br), heart (He), lung (Lu), liver (Li), kidney (Ki), spleen (Sp), female genital tract (Fgt) and testis (Te).

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3.3.1. Sertoli cells of the testis Light microscopy of adult testis sections treated with the anti Stra6 antibody revealed a strong positive staining restricted to the basal layer of the seminiferous epithelium (Fig. 4A). The intensity of staining showed important variations between different tubules, indicating that Stra6 expression depends on the stage of the spermatogenic

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cycle. Histological examination revealed that Stra6 expression was restricted to stage VI and VII tubules (Russell et al., 1990). Interestingly, immunoreactions performed on serial testis sections showed that Stra6 was expressed in the same tubules as those expressing Stra8 (Fig. 4A,B), another RA-inducible gene of unknown function, but whose expression was shown to be restricted to premeiotic

Fig. 4. Testis expression of Stra6 and Stra8 proteins. Sections (10 mm) from wild-type (A,B), RXRb null (C,D) and RARa null (E, F) mutant testes were processed according to the ABC immunoperoxydase method with purified polyclonal antibodies against Stra6 (A,C,E) or Stra8 (B,D,F) proteins. Note the colocalization of both proteins in the same seminiferous tubules, and the abnormally high proportion of labeled tubules in RARa mutant testis. Sections were slightly counterstained with methyl green. Bar, 100 mm.

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3.3.2. Retinal pigment epithelium Both immunohistochemical and ISH analyses revealed a strong expression of Stra6 in the eye and the periocular mesenchyme during development. At 9.5 days post-coitum (d.p.c.), Stra6 was expressed in the optic vesicle (data not shown). From 10.5 d.p.c., Stra6 transcripts were found in the retinal pigment epithelium (RPE), the inner nuclear layer of the neural retina, the anteriormost part of the lens, the meninges of the optic nerve and the periocular mesenchyme (Fig. 6A; see also Fig. 8E,F and Fig. 10D; and data not shown). No transcripts were detected in the developing cornea. At 16.5 and 17.5 d.p.c., after the closure of the eyelids, Stra6 was also expressed in the epithelium lining the conjunctival sac (Fig. 6A). In the adult eye, Stra6 protein remained expressed only in the RPE and the meninges surrounding the optic nerve (Fig. 6B; and data not shown).

Fig. 5. Stra6 subcellular localization in Sertoli cells. Immunohistochemistry was performed with a purified anti-Stra6 antibody on 100 mm sections of adult mouse testis, without counterstaining. Sections (70 nm) were then cut and observed by light microscopy (A) or electron microscopy (B). Dark immunoperoxydase staining was restricted to the basal pole of Sertoli cell plasma membrane. Note the absence of staining of the plasma membrane of spermatogonia (open arrows). c, cytoplasm; n, nucleus; pl, plasma membrane of Sertoli cell; S, Sertoli cell; Sg, spermatogonia; Sp, spermatocyte. Bars, 12 mm (A), 1 mm (B).

germ cells (Oulad-Abdelghani et al., 1996). Since RARa and RXRb null mutants which have been generated in our laboratory (Lufkin et al., 1993; Kastner et al., 1996) are sterile and exhibit abnormalities in the spermatogenesis process, we analyzed Stra6 and Stra8 expression in the testes of these mutants. No alteration in Stra6 or Stra8 protein expression could be detected in the testes of RXRb−/− mutants (Fig. 4C,D). In contrast, RARa−/− mutant testes displayed abnormal distributions of Stra6 and Stra8 proteins, both of them being expressed in almost all the tubules (Fig. 4E,F). To precisely determine the cell type expressing the Stra6 protein, as well as its subcellular localization, we performed immuno-electron microscopy on thin testis sections. Stra6 was found to be localized in the plasma membrane of the basal pole of Sertoli cells, whereas the plasma membrane of neighboring spermatogonia was devoid of this protein (Fig. 5; and data not shown).

3.3.3. Choroid plexi and brain microvasculature Immunohistochemistry and ISH were also used to analyze the expression of Stra6 in the developing and adult central nervous system. Stra6 transcripts were detected in the developing meninges (e.g. Fig. 6A and Fig. 7A–D), in the choroid plexi and in part of the microvasculature from 11.5 d.p.c. to birth (Fig. 7A–D; and data not shown). Labeling of the microvessels of the striatum (Fig. 7A–D) and of the spinal cord (Fig. 10C,E,F) was particularly strong during fetal life. The strong and patchy labeling seen in the developing cranial and dorsal root ganglia (Fig. 10B,D), as well as in the sympathetic chain ganglia (Fig. 10E,F), may also be related to the microvasculature. Note that Stra6 was transiently expressed in other neural tissues during embryonic development (see below). Immunohistochemical stainings of mouse fetuses were consistent with the ISH results (data not shown). In adult mice, both Stra6 transcripts (not shown) and protein were detected in the choroid plexi (Fig. 7E), in brain microvessels (Fig. 7F) and in the meninges (data not shown). Interestingly, Stra6-expressing capillaries were not broadly distributed throughout the brain, but were mainly located in the striatum, the preoptic zone, the medial cerebellar and ventral cochlear nuclei (Fig. 7F; and data not shown). Thus, only a small subset of the brain microvasculature was found to express Stra6. However, Stra6 protein or RNA were not detected in the microvascular endothelium of any of the other adult organs that were examined in similar immunohistochemical and ISH assays. Neuronal and glial cells did not express Stra6 in the adult (data not shown). 3.3.4. Yolk sac and chorioallantoic placenta At 6.5 and 7.5 d.p.c., Stra6 was strongly expressed in the innermost cells of the uterine wall, encircling the entire implantation site, as well as in the primitive endoderm, which will later give rise to the yolk sac (Fig. 8A; and data not shown). From 8.5 to 9.5 d.p.c., the decidua, yolk sac membrane (or primitive placenta), and chorionic zone (future chorioallantoic placenta) expressed Stra6 transcripts

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Fig. 8. Expression of Stra6 transcripts in the yolk sac and placenta. Histological sections of mouse conceptuses were hybridized to an antisense Stra6 riboprobe. Developmental stages shown are 6.5 d.p.c. (A,B), 9.5 d.p.c. (C,D) and 12.5 d.p.c. (E–H). Panels A, C, E and G are dark-field views showing the signal grain in white and panels B, D, F and H are bright-field views of the same sections revealing the histology. C, chorionic plate; D, decidua; E, embryo; F. fetus; GC, trophoblastic giant cell; Lb, labyrinth; P, placenta; Sp, spongiotrophoblast; U, uterus; YS, yolk sac.

(Fig. 8C). However, the decidual labeling persisted only in the antimesometrial and mesometrial poles (Fig. 8C). From 9.5 to 11.5 d.p.c., Stra6 expression increased in the developing chorioallantoic placenta and decreased in the yolk sac membrane. At 12.5 d.p.c., strong expression of Stra6 was seen in the labyrinthine region of the placenta, which is the zone of exchange between maternal and fetal bloods (Fig. 8E–G). At the same stage, Stra6 expression was no longer detected in the yolk sac membrane. Weak labeling persisted in the decidua up to 13.5 d.p.c. (data not shown). During mid-late placentation stages (13.5 d.p.c. to birth), Stra6 expression was restricted to the labyrinthine zone of the chor-

ioallantoic placenta (data not shown). The trophoblastic giant cells never expressed detectable levels of Stra6 transcripts. 3.4. Stra6 expression in other adult organs Immunohistochemistry and ISH were also used to determine the sites of expression of Stra6 protein and transcripts in several adult organs. In the female genital tract, the protein was found to be strongly expressed in the endometrium and in the granulosa cells surrounding ovarian growing follicles (data not shown). Stra6 transcripts and proteins were

Fig. 6. Immunohistochemical localization of Stra6 protein in the eye during development and in adult CD1 mice. (A) Transverse section through the head of a 17.5 d.p.c. fetus. (B) Section of the retina of an adult mouse. br, brain; c, cornea; el, eyelid; l, lens; m, meninges; nr, neural retina; on, optic nerve; rpe, retinal pigment epithelium. Fig. 7. Stra6 transcript and protein expression in the meninges, cranial ganglia, choroid plexi and brain microvasculature. (A,B) Parasagittal section through the head of a 16.5 d.p.c. fetus hybridized to an antisense Stra6 riboprobe (bright-field and dark-field views, respectively), showing expression in the meninges, choroid plexi, trigeminal ganglion and striatum microvasculature. (C,D) Enlargement of the striatal region from the same section (boxed in A). (E,F) Immunoreactive Stra6 protein, indicated by the brown reaction product, is detected in the choroid plexus (E) and brain microvessels (F) of an adult mouse. Labeling in the choroid plexi corresponds to the epithelium. However, we cannot exclude that there is also some Stra6 expression in the endothelium. co, cochlea; cp, choroid plexi; m, meninges; nc, nasal cavity; on, optic nerve; st, striatum; tb, tooth bud; tg, trigeminal ganglion.

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Summary of Stra6 expression sites during mouse development. Note that Stra6 expression features in the developing limbs have been described elsewhere (Chazaud et al., 1996) System/organ Nervous system

Face

Ear Eye

Muscle Skeletal system Embryonic gut derivatives Urinary system Genital system

a

Stra6 expression sites

Stages (d.p.c.) a

See figure

Basal plate of the spinal cord Fore and midbrain Mid-hindbrain boundary Infundibulum, neurohypophysis Frontonasal mesenchyme, nasal epithelium and mesenchyme Mandibular and maxillary mesenchyme Otocyst and inner ear epithelium External ear mesenchyme Retina, inner nuclear layer Retinal pigment epithelium Lens Periocular mesenchyme Somites, myotomes Body and head muscles Around chondrogenic condensations, perichondrium

8.5–16.5 9.5 8.5–16.5a 11.5 to adult 9.5–16.5a

9E and 10C 10A 10A 10C 7A, 10A–C

9.5–11.5b 9.5–16.5a 13.5–16.5a 9.5 to adult 10.5 to adult 9.5 to adult 9.5–13.5 8.5–12.5b 13.5–16.5a 13.5–16.5a

10A–C 10A,B,D

Pharyngeal pouches, thymus Esophagus, lung, stomach, intestine and rectum mesenchymes Intermediate mesoderm, mesonephros Metanephros, kidney, urethra Female genital tract, vas deferens Ovary Testis

9.5–16.5a 9.5–16.5a 8.5–l2.5b 12.5 to adult 15.5 to adult 13.5 to adult 15.5 to adult

10D 6A,B 6A 10D 9C–F, 10A,C,D and Chazaud et al., 1996 10D and Chazaud et al., 1996 10A 10A,C,F 9E,F and 10C 10D,F 8A,B and 10F 4 and 5

Stra6 expression may persist at later stages. Stra6 expression is seen later in derivatives of these structures.

b

also found in spleen, in the bronchial epithelium, in the convoluted tubules of the kidney, in sparse cells of the thymic medulla and in the neurohypophysis (data not shown). 3.5. Developmental expression of Stra6 We have previously reported the expression pattern of Stra6 in developing limbs and during endochondral ossification (Chazaud et al., 1996). Hereafter, we describe additional developmental expression features revealed by ISH analyses. These data are summarized in Table 1. 3.5.1. Stra6 expression at early developmental stages By 6.5 d.p.c., no hybridization signal was seen in the embryo proper (Fig. 8A). During gastrulation (7.5 d.p.c.), the gene was expressed in the posterior mesoderm (Fig. 9A,B), the headfold mesoderm being unlabelled (Fig. 9A). Interestingly, Stra6 transcripts were not detected in the primitive streak, but in more lateral regions of the mesoderm (Fig. 9B). Stra6 was expressed in the neural plate and in various mesodermal derivatives by 8.5 d.p.c. (Fig. 9C). In caudal regions, Stra6 was evenly expressed in the neural plate and the presomitic mesoderm (Fig. 9D). Cranially, in more differentiated regions, Stra6 expression was specific of the basal plate of the neural tube and was more pronounced in the dorso-medial part of the developing

somites (Fig. 9E,F). In addition, Stra6 was expressed in the intermediate mesoderm, at the level of the pronephric duct (Fig. 9E,F; and data not shown), as well as in the dorsal parts of the somatic and splanchnic mesoderm layers bordering the coelomic cavity (Fig. 9E,F). 3.5.2. Stra6 expression in the differentiating nervous system At 9.5 d.p.c., Stra6 was expressed at the level of the midbrain-hindbrain boundary and more weakly in the midbrain and forebrain neuroepithelium (Fig. 10A). At later stages, certain neural structures were labeled, in addition to the aforementioned brain and spinal cord microvasculature. Labeling was seen in the infundibulum by 11.5 d.p.c., and later in the developing neurohypophysis (Fig. 10C; and data not shown). From 11.5 to 16.5 d.p.c., the transcripts were seen in the developing cerebellum (data not shown). Stra6 expression persisted until at least 15.5 d.p.c. in the basal plate of the spinal cord mantle layer (open arrow in Fig. 10C; and data not shown). 3.5.3. Stra6 expression in sensory organs Expression of Stra6 was detected in three developing sensory organs: the eye (see above), the nose and the ear. Stra6 expression was already present in the frontonasal mesenchyme at 9.5 d.p.c. (Fig. 10A; and data not shown). By 11.5–12.5 d.p.c., strong labeling was seen around the

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Fig. 9. Expression of Stra6 transcripts in 7.5 and 8.5 d.p.c. embryos. (A) Whole-mount in situ hybridization of a 7.5 d.p.c. embryo. (B) Transverse section of the same embryo, showing labeling in the mesodermal and endodermal layers. The approximate plane of sectioning is indicated by thick arrows in (A). (C) Whole-mount in situ hybridization of an 8.5 d.p.c. (~8 somites) embryo. (D–F) Caudal to rostral transverse sections of the same embryo. The approximate planes of sectioning are indicated in (C). Note the labeling of the presomitic and somitic, intermediate and lateral mesoderm, as well as the distinct labeling patterns along the neural tube. A-P, antero-posterior axis; ao, aorta; b, brain; co, coelomic cavity; en, endoderm; et, extraembryonic tissues; hb, hindbrain; hf, headfolds; im, intermediate mesoderm; l, lateral part of the somite; m, medial part of the somite; me, mesoderm; n, notochord; ne, neuroectoderm; np, neural plate; nt, neural tube; pnt, pronephric tubule; ps, primitive streak; psm, presomitic mesoderm; s, somite; so, somatopleure; sp, splanchnopleure.

nasal cavities (Fig. 10B,C). At 15.5–16.5 d.p.c., Stra6 expression was restricted to the olfactory and respiratory mesenchymes and to parts of the respiratory epithelium (Fig. 7A,B). By 9.5 d.p.c., the gene was expressed in the

otocyst epithelium (Fig. 10A). At later stages, expression was seen in the epithelium of the differentiating inner ear, as well as in the external ear mesenchyme (Fig. 10B and Fig. 7A; and data not shown).

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Fig. 10. Developmental expression of Stra6 from 9.5 to 16.5 d.p.c.. (A) Sagittal section of a 9.5 d.p.c. embryo. The open arrow shows the expression in the first pharyngeal arch mesenchyme. (B) Parasagittal section of an 11.5 d.p.c. embryo. (C) Sagittal section of a 12.5 d.p.c. embryo. The open arrow points to the labeling in the basal plate of the spinal cord. (D) Parasagittal section of a 13.5 d.p.c. embryo. (E,F) Enlargement of the abdominal region of a 16.5 d.p.c. female fetus (bright-field and dark-field views, respectively). ao, aorta; b, bladder; bm, brain microvasculature; cp, choroid plexus; drg, dorsal root ganglia; e, eye; ea, inner ear; fl, forelimb; fb, forebrain; g, gut; gb, genital bud; hlc, hindlimb cartilages; i, intestine; in, infundibulum; k, kidney; l, lung; m, meninges; me, mesonephros; mhb, mid-hindbrain boundary; mu, muscles; nc, nasal cavities; o, otocyst; oe, esophagus; pp, pharyngeal pouches; pv, prevertebrae; r, rib; re, rectum; s, stomach; sc, spinal cord; sg, sympathetic ganglion; so, somite; t, tongue; tg, trigeminal ganglion; u, urethra; va, vagina.

3.5.4. Stra6 expression in embryonic gut derivatives By 9.5 d.p.c., transcripts were detected in the central mesenchyme of the mandibular arch and in the epithelium of the pharyngeal pouches (Fig. 10A). The thymus, which is a derivative of these pouches, was labeled at later stages of development (data not shown). At 11.5 d.p.c. and later stages, Stra6 was strongly expressed in the mesenchyme of the esophagus, stomach, intestine and rectum (Fig. 10B,C,E,F; and data not shown). The submandibular salivary glands,

which arise from an invagination of the endoderm of the oral cavity, also expressed Stra6 (data not shown). Restricted signals were also seen in the mesenchyme of the developing tooth buds (Fig. 7A,B). The lungs, that develop from a foregut diverticulum, were labeled at the level of the mesenchyme surrounding the bronchi (Fig. 10D). 3.5.5. Stra6 expression in the excretory and genital tracts Stra6 expression was also detected in differentiating

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nephric tissues. The gene was first expressed in the mesonephric mesenchyme, excluding the genital ridges (Fig. 10C; and data not shown), and then in the definitive kidney (Fig. 10D). Patchy labeling was seen in the renal cortex as well as in the developing metanephric collecting tubules (Fig. 10D). Hybridization signals were also detected in the ureteric, urethral and genital bud mesenchymes (Fig. 10C, E,F). By 16.5 d.p.c., in the female genital tract, both the epithelia and mesenchyme of the oviduct and uterus were strongly labeled, whereas only the epithelium of the vagina showed some hybridization signal (Fig. 10E,F; and data not shown). Patchy expression was seen in the ovary (data not shown). In the male genital tract, Stra6 was expressed in the vas deferens and in all the seminiferous tubules of the fetal testes (data not shown). 3.5.6. Stra6 expression in developing muscles and skeleton By 11.5 d.p.c., Stra6 was specifically expressed in the myotomes, as well as in the myogenic cells of the limb buds (Chazaud et al., 1996; and data not shown). At later stages, expression was found in the various developing skeletal muscles of the body and face (e.g. Fig. 10D). At 12.5 d.p.c., Stra6 was expressed in the mesenchyme surrounding the various chondrogenic condensations (e.g. Fig. 10B,C, around the prevertebrae). By 13.5 d.p.c., the expression was restricted to the perichondrium (e.g. Fig. 10D, around the ribs and the hindlimb cartilages). Later (16.5 d.p.c.), Stra6 transcripts were further limited to the perichondrium surrounding the ossification centers (Chazaud et al., 1996).

4. Discussion We have previously reported the isolation of several cDNA fragments corresponding to RA-inducible genes in P19 embryonal carcinoma cells (Bouillet et al., 1995). A 3 kb cDNA containing a 2010 nt long ORF was isolated, using one of these cDNA fragments (Stra6) as a probe. Computer analysis of the protein sequence deduced from this ORF predicts a highly hydrophobic protein harboring nine putative transmembrane domains. As already described for a number of membrane proteins such as nicotinic acetylcholine receptor, b-adrenergic receptor, rhodopsin and lactose permease (Thomas and McNamee, 1990), the Stra6 protein aggregated when boiled in Laemmli protein sample buffer, thus suggesting that it could be an integral membrane protein. We have shown by electron microscopy that Stra6 protein is indeed localized to the plasma membrane in Sertoli cells. Despite these characteristics, comparison of the Stra6 sequence with proteins of databases did not reveal any significant similarity with well-known integral membrane proteins such as rhodopsin, neurotransmitter receptors, transporters or ion channels. Thus, Stra6 appears to belong to a new type of membrane proteins. Interestingly, the Stra6 gene has been conserved during evolution, since the mouse

probe detected a bovine RNA in a high stringency Northern blot experiment. Stra6 was initially isolated as an RA-inducible gene in P19 embryonal carcinoma cells (Bouillet et al., 1995). We have shown that it is also induced by T-RA in F9 and ES cells, whose differentiation is also induced by RA. As some of the other RA-inducible genes isolated in the same screening experiment (e.g. GAP 43, Stra7 or Stra11) were not induced in all three cell lines (Bouillet et al., 1995), the expression of Stra6 may be involved in differentiation processes. Stra6 was used in our laboratory as a marker to study the consequences of the inactivation of retinoid receptor genes in F9 cells. Stra6 inducibility was compared in WT, RARa−/−, RARg−/−, RXRa−/−, RXRa−/−/RARa−/− and RXRa−/−/RARg−/− cells (Taneja et al., 1995; Chiba et al., 1997). The RA induction of Stra6 was shown to be preferentially mediated by RXRa/RARg heterodimers, since inactivation of either one of these genes led to an important decrease of this induction. On the other hand, its expression was found to be consistently increased in RA-treated RARa−/− cells, indicating that RARa may repress Stra6 transcription (Taneja et al., 1995; Chiba et al, 1997). These data, together with the rapid induction of the gene after RA addition, indicate that Stra6 is probably a direct target of the retinoid receptors. In vivo, RARa and Stra6 are coexpressed in Sertoli cells. Interestingly, we show that inactivation (gene knockout) of RARa in the mouse leads to a widespread expression of Stra6 in most seminiferous tubules of the testes, as if RARa would normally repress the expression of Stra6 at certain stages of the spermatogenic cycle. Thus, a negative effect of RARa on Stra6 expression has been observed in two very different instances. It will be interesting to characterize the cis-elements controlling the activity of the Stra6 promoter, in order to elucidate the molecular mechanisms involved in these inductive and repressive effects. We also observed that two RA-responsive genes, Stra6 and Stra8, were co-expressed in the same testis seminiferous tubules, i.e. at the same stages of the spermatogenic process, but in distinct cell types (Sertoli cells and premeiotic germ cells, respectively). This may reflect the importance of somaticgerm cell interactions in the regulation of spermatogenesis. For instance, Desert hedgehog (Dhh) is a secreted molecule expressed by Sertoli cells, and inactivation of the Dhh gene results in a marked germ cell deficiency (Bitgood et al., 1996). Targeted disruptions of the Stra6 and Stra8 genes may provide additional information concerning cell-to-cell interactions in spermatogenesis and the role of retinoids in this process. Using both ISH and immunohistochemical techniques, high expression of Stra6 was demonstrated in blood-organ barriers. These barriers are made of epithelial or endothelial cells associated by tight junctions. Their role in limiting passive diffusion of chemicals, as well as their function in the facilitative transport of many nutrients, have been well

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documented. For example, specific transport systems for thiamine, ascorbic acid, pyridoxine, folate and inositol have been demonstrated in the cuboidal cells of the choroid plexi (Pardridge, 1986a, 1986b). Glucose transport through the blood-brain barrier is mediated by a protein, GLUT-1, which is asymmetrically localized on luminal and adluminal membranes of brain endothelium (Pardridge, 1991). The presence of Stra6 in different cell types of distinct blood-organ barriers suggests that it may perform the same function in these cells. The high expression of Stra6 in these 16 barriers, together with its localization in the basal region of the Sertoli cell plasma membrane, makes it a good candidate as a component of such a transport system.

Acknowledgements We are grateful to Dr M. Mark for useful comments, S. Heyberger and S. Bronner for excellent technical assistance, S. Vicaire for DNA sequencing work, P. Eberling, D. Queuche and A. Staub for peptide synthesis, F. Ruffenach, I. Colas, P. Hamannn and E. Troesch for oligonucleotide synthesis, J.M. Lafontaine and B. Boulay for artwork, G. Duval for antibody production and J.L. Vonesch for technical advice. This work was supported by funds from the Institut National de la Sante´ et de la Recherche Me´dicale, the Centre National de la Recherche Scientifique, The Centre Hospitalier Universitaire Re´gional, the Association pour la Recherche sur le Cancer (ARC), the Ministe`re de la Recherche et de l’Espace (grants 92HO932 and 92N60/0694), the Fondation pour la Recherche Me´dicale (FRM), the Colle`ge de France and Bristol-Myers Squibb. P.B. is a recipient of a fellowship from the ARC. V.S. is on leave from the INSERM U 384 (Clermont-Ferrand, France).

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