SEARCH

SEARCH BY CITATION

Keywords:

  • Alex2;
  • armadillo;
  • gonadal development;
  • sex determination;
  • testis;
  • interstitial cells

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. REFERENCES

In a screen for transcripts differentially expressed during gonadal development in mouse embryos, we identified the novel armadillo-related gene, Alex2. The armadillo (arm) family of proteins share a 42 amino acid tandem repeat motif called the arm domain, through which they interact with different binding partners. These intracellular proteins are implicated in a variety of developmental processes, including cell proliferation, migration, maintenance of tissue integrity, and tumorigenesis. Alex2 is a member of a novel subgroup within the arm family, encoding a protein with a single arm domain and a putative transmembrane or signal sequence. Alex2 has a developmentally regulated expression profile during embryogenesis in the mouse. In the urogenital system, it is strongly expressed in the developing testis but is down-regulated during ovarian development. Alex2 expression is localized within the interstitial cell lineage of the developing testis, which gives rise to peritubular myoid, endothelial, and fetal Leydig cells. Alex2 is also expressed in the developing forebrain and somites and in dorsal root ganglia. In testicular cell lines, Alex2 fusion proteins localize to membrane structures within the cell. The expression profile of Alex2 suggests that it plays a role in the development of several tissues during embryogenesis, notably testicular differentiation. In the developing testis, its expression profile suggests that Alex2 has a role in specification or development of the interstitial cell lineage. Developmental Dynamics 233:188–193, 2005. © 2005 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. REFERENCES

The adult mammalian testis has two important functions: production of gametes (sperm) and hormone secretion. The structure of the testis reflects these two functions. It is a compartmentalized organ with seminiferous tubules dedicated to sperm production, surrounded by interstitial cells concerned largely with androgen biosynthesis. Testis structure is established during embryogenesis, when the Y-linked Sry gene is expressed in male (XY) embryos. In the mouse, Sry is expressed transiently in the supporting pre-Sertoli cell lineage over embryonic day (E) 11.5–12.5. Sry expression initiates a cascade of molecular and cellular events leading to testicular differentiation (Brennan and Capel,2004). Sex reversal syndromes in humans and targeted mutagenesis in mice have led to the identification of several genes downstream of Sry that are also important for morphogenesis of the testis (reviewed in Swain and Lovell-Badge,1999). To identify additional genes involved in mammalian gonadal development, we conducted a subtractive hybridization screen using male versus female embryonic mouse gonads (McClive et al.,2003). One gene isolated from this screen and enriched male-specifically was Alex2 (Armadillo protein Lost in Epithelial cancers, on chromosome X, #2). Alex2 (also called Armcx2) is the murine ortholog of a novel human gene found to be down-regulated in a variety of carcinomas (Kurochkin et al.,2001).

Alex2 encodes a novel member of the armadillo family of proteins. The founding member, armadillo, is a Drosophila segment polarity gene required for wingless signal transduction (Wieschaus and Riggleman,1987). Armadillo proteins share a 42 amino acid tandem repeat motif called the arm domain, through which they interact with different binding partners (Peifer et al.,1994). They are intracellular proteins and are implicated in a variety of developmental processes, including cell proliferation, migration, maintenance of tissue integrity, and tumorigenesis. Armadillo proteins have dual roles, combining both structural and signal transduction functions (reviewed in Hatzfeld,1999). Some important and well-studied members of the armadillo family include β-catenin, the nuclear import factor importin, and the tumor suppressor adenoma polyposis coli (APC). β-Catenin transduces the Wnt signaling pathway within the cell. Stabilized β-catenin regulates gene expression through interaction with the TCF/LEF transcription factor complex (the so-called “canonical” pathway). Wnt signal transduction through β-catenin is an essential developmental process, important for cell fate specification in many tissues. β-catenin is also involved in cell–cell adhesion (binding E-cadherin). In the context of gonadal development, β-catenin and the signaling molecule Wnt4 are both implicated in testicular differentiation, but antagonistically. β-Catenin has a stimulatory effect on developing testicular Leydig cells, whereas Wnt4 antagonizes this effect (Jordan et al.,2003).

Bioinformatic analysis shows that the conceptual mouse Alex2 protein is one of three related sequences: Alex1, 2, and 3 (Fig. 1). These proteins share approximately 60% sequence similarity and are encoded by three separate but closely linked genes on the X chromosome. Mouse Alex2 encodes a predicted protein of 782 amino acids, including a 300 amino acid internal region that is absent in mAlex1 and mAlex3 (Fig. 1). (Of interest, this region is partially conserved in rat but is absent in human ALEX2.) The three Alex genes have presumably evolved through duplication of a single ancestral gene. Together, they represent an unusual subgroup within the armadillo family, because they have only one or two arm domains (all other family members have at least six), and they have a conserved hydrophobic N terminus predicted to specify a transmembrane domain or signal sequence, potentially targeting them to the secretory pathway of the cell. All other armadillo proteins lack signal sequences. The genomic organization of Alex2 is conserved between human and mouse, with the entire 2.35-kb open reading frame being encoded by the last of six exons. Analysis of the expressed sequence tag (EST) database indicates that some shuffling occurs among the five upstream exons, but the last exon encoding the full protein is always included.

thumbnail image

Figure 1. Alignment of the deduced mouse Alex1, Alex2, and Alex3 proteins. The putative transmembrane/signal sequence is underlined in black, and the armadillo domain is contained in the boxed area. The highest homology between these three proteins occurs at the carboxy terminus. Mouse Alex2 includes a 300 amino acid internal region that is absent in the other two proteins. Accession numbers: mouse Alex2, NM_026139; mouse Alex1, NM_030066; mouse Alex3, NM_027870.

Download figure to PowerPoint

The Alex proteins are highly conserved between mouse and human (95% amino acid similarity for Alex1, 80% for Alex2, and 98% for Alex3). This finding suggests that these novel proteins have critical functions. In the only other study that has been published on the ALEX genes, Kurochkin and colleagues (2001) found that both ALEX1 and ALEX2 mRNAs are widely expressed in adult human tissues (embryonic expression was not examined). Intriguingly, they found that expression of both genes is lost in carcinomas (cancers of epithelial origin), including adult ovarian, colon, and prostate cancers. Altogether, this preliminary data suggests that ALEX proteins may normally act as tumor suppressors, being required to maintain tissue integrity and/or prevent cell hyperproliferation. Apart from Unigene EST data (Mm.285969), mouse Alex2 expression has not been reported previously. This study describes the expression profile of mouse Alex2 during embryogenesis, with particular emphasis on testicular differentiation.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. REFERENCES

Alex2 mRNA expression was examined during gonadogenesis in the mouse embryo, using whole-mount in situ hybridization (Fig. 2). In the mouse, the gonad is morphologically undifferentiated until E11.5, when Sry expression peaks in males and testicular differentiation commences. Sry is expressed in pre-Sertoli cells, which organize into seminiferous cords. In embryos examined at embryonic day (E) 11.5, weak Alex2 expression was detected at the boundary between the mesonephric (embryonic) kidney and undifferentiated gonad of both sexes (Fig. 2A,F). At E12.5, the onset of morphological differentiation of the testis, Alex2 was strongly expressed in a network pattern in male gonads (Fig. 2B). In contrast, from E12.5, expression became undetectable in females by whole-mount in situ staining (Fig. 2G). In the developing testis, strong regionalized expression was maintained until E16.5, when expression declined (Fig. 2C–E). The reticulate pattern of staining in developing male gonads suggests expression of Alex2 in the interstitial cell population. These cells differentiate around the seminiferous cords and are separated from the cords by basement membrane. They give rise to fetal Leydig, endothelial, and peritubular myoid cells. To verify expression of Alex2 specifically in the interstitial cells, E12.5 and 13.5 whole-mount stained male gonads were cryosectioned. Transverse sections confirmed widespread Alex2 mRNA expression in the interstitial cell population at both time points (Fig. 2K). Immunostaining of E13.5 male gonads after in situ hybridization using cell type-specific markers confirmed Alex2 expression in the interstitial cell lineage. Expression of the Sertoli cell marker AMH delineated the developing seminiferous cords, which were negative for Alex2 expression (Fig. 2L).

thumbnail image

Figure 2. Expression profile of Alex2 during embryogenesis in the mouse, assessed by whole-mount in situ hybridization. A–G: Gonadal expression. H–J: Whole embryo expression. K–O: Gonadal tissue sections. A: In male embryonic urogenital systems, Alex2 mRNA expression is first detectable between the gonad (G) and mesonephric kidney (Ms) at embryonic day (E)11.5. B,C: Strong gonad-specific expression (arrowheads) is detectable in the differentiating testis (T) at E12.5 (B) and E13.5 (C). D,E: Expression persists at E14.5 (D) but is undetectable by E16.5 (E). F,G: In female embryos, a similar expression pattern is detectable at the gonad–mesonephros interface at E11.5 (F), whereas expression becomes undetectable from E12.5 (G). H: In whole embryos examined at E9.5, Alex2 is also expressed in the forebrain (arrowhead) and somites (arrow) of both sexes. I: At E10.5, brain and somitic expression persists. J: By E11.5, strong expression is seen in the region of the dorsal root ganglia (arrowheads) and in the developing ribs (arrow). K: In sections taken from E13.5 whole-mount-stained testes, Alex2 expression is localized in the interstitial compartment (I in K) and is not detectable in the developing seminiferous cords (Sc). L: Immunohistochemical localization of the Sertoli cell marker protein, Amh (brown, Sc), showing mutually exclusive expression with that of Alex2 mRNA (blue, I in L). M–O: Expression of other cell-type markers in the E13.5 mouse testis. M: interstitial cell expression (I in M) of the neurotrophin receptor protein p75NTR. N: Immunofluorescent detection of the Sertoli cell marker Sox9 (red) and the endothelial and germ cell marker PECAM (green, shown by white arrow). O: Laminin expression (green) outlines the seminiferous cords and delineates the interstitium. Scale bars in = 100 μm in A–G,K–M (applies to A–G,K–O), 0.5 mm in H–J.

Download figure to PowerPoint

In contrast, the neurotrophin receptor protein p75NTR, a marker of interstitial cells, showed coincident spatial expression with that of Alex2 (Fig. 2M). The p75NTR protein identifies mesenchymal interstitial cells in the early mouse testis (up to E12.5), becoming restricted to the peritubular myoid cells as differentiation proceeds (Campagnolo et al.,2001). Like p75NTR, Alex2 appears to be expressed in the pluripotent progenitor population of mesenchymal cells (compare Fig. 2K,M). Alex2 may, therefore, be involved in the early phases of interstitial cell specification or function in the developing testis. By E14.5 in the mouse, the interstitial cells have given rise to peritubular myoid, vascular endothelial, and steroidogenic Leydig cells (Fig. 2N). At this time, Alex2 did not become restricted but was still widely expressed throughout the interstitium (data not shown). This finding suggests that Alex2 continues to play a role in all of these cell types. However, by E16.5, no gonadal expression could be detected by whole-mount in situ hybridization (Fig. 2E).

Outside the urogenital system, Alex2 expression was also detected in the nervous system and in developing somites. In E9.5 embryos of both sexes, expression was observed in the forebrain and midbrain and in the somitic mesoderm (Fig. 2H). This expression pattern persisted in E10.5 embryos (Fig. 2I). By E11.5, expression was evident in the region of the dorsal root ganglia and in early forming ribs (Fig. 2J). These extragonadal sites of expression were not sexually dimorphic.

Gonadal Alex2 expression was also studied by reverse transcriptase-polymerase chain reaction (RT-PCR) over E11.5–E16.5. Due to the small size of tissues, gonads+mesonephric kidneys were excised at E11.5. For all other stages, the gonads alone were isolated. PCR was carried out at the linear phase of amplification. Ethidium-stained bands were semiquantitated and standardized against Hprt expression. Using this method, Alex2 expression was detected in both sexes over developmental times (Fig. 3A,B). At E11.5, expression was similar in males and females, as suggested by the whole-mount staining. Expression was subsequently maintained in male gonads, declining later at E16.5. In females, expression declined sharply after E11.5 (Fig. 3B). The detection of Alex2 expression in females by RT-PCR but not by whole-mount in situ hybridization could be explained by the greater sensitivity of the former method. (For both the RT-PCR primers and whole-mount riboprobes, specific Alex2 sequences were used, excluding potential cross-hybridization with Alex1 or Alex3.) Alex2 expression was also detectable by RT-PCR in adult mouse testis and ovary, and in kidney, liver, and brain (data not shown).

thumbnail image

Figure 3. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of mAlex2 expression in developing mouse gonads. A: Expression of Alex2 is detectable in both sexes after 22 cycles of amplification. F, female; M, male. RT−, reverse transcription of embryonic day (E) 13.5 male gonad in the absence of reverse transcriptase (negative control). Hprt-positive control is shown for each sample. All samples represent several pooled gonad pairs, except for E11.5 time points, which comprise gonads plus mesonephric kidneys. B: Semiquantitative analysis of Alex2 gene expression in male (closed circles) and female (open circles) embryonic gonads during the linear phase of PCR amplification (22 PCR cycles). Expression is initially similar in both sexes but becomes down-regulated in females from E12.5. In males, maximal expression occurs in the early phases of testicular differentiation (E11.5–E14.5) and declines thereafter.

Download figure to PowerPoint

The sexually dimorphic expression pattern of Alex2 in embryonic mouse gonads may be related to the male-specific migration of mesenchymal cells. In vitro organ culture experiments have shown that mesenchymal cells move into the gonad from the neighboring mesonephros early in testis formation (from E11.5; Buehr et al.,1993; Martineau et al.,1997). This process is essential for seminiferous cord organization in males and it does not occur in female (XX) gonads (Tilmann and Capel,1999). The cells enter the gonad in response to Sry expression and pre-Sertoli cell differentiation (Capel et al.,1999). Immigrating cells contribute to the interstitial cell population, namely, myoid and endothelial cells, but probably also fetal Leydig cells. The myoid cell precursors in particular cooperate with Sertoli cells in organizing the seminiferous cords. As Alex2 is expressed in the region between the gonad and mesonephros at E11.5 (in both sexes), it may play a role in the competency of those cells destined to migrate into the gonad. By E12.5, after Sry activation, the maintenance of Alex2 expression in males may represent those cells migrating into the gonad and proliferating. In contrast, no Sry expression occurs in the female (XX) gonad, no immigration of cells occurs and Alex2 expression declines. Interestingly, it has been shown that neurotrophin signaling from the developing Sertoli cells plays a role in the induction of mesonephric cell migration (Cupp et al.,2003), and Alex2 is expressed in the same gonadal cells as the neurotrophin receptor p75NTR (Fig. 2M). Taken together, these observations suggest that Alex2 may play a role in the key mesenchymal cell migration process underlying testis development.

To examine the subcellular localization of Alex2 protein, the 2.35-kb open reading frame was isolated from E12.5 mouse testis cDNA and cloned into a FLAG-tagged expression vector. After transfection into the testicular Leydig cell line TM3, subcellular localization was assessed using a FLAG antibody. At 24 hr after transfection, Alex2 epitope-tagged protein could be detected in discreet membrane structures within the cell (Fig. 4A). This localization indicates that Alex2 has a membrane-associated function or that it is targeted to the secretory pathway of the cell. This finding is consistent with the predicted transmembrane/signal sequence located at the amino terminus of the protein (Fig. 1). Antibody markers used to identify subcellular organelles suggest that Alex2 is not specifically localized within either endoplasmic reticulum or the perinuclear Golgi apparatus (Fig. 4B,C). If Alex2 is indeed secreted, it may play a role in extracellular signalling during testicular differentiation. Potential secretion of Alex2 protein into cell culture medium is currently under examination. Based on its embryonic expression profile, it is hypothesized that Alex2 has a developmentally regulated function during mouse embryogenesis, including testicular differentiation.

thumbnail image

Figure 4. Subcellular localization of FLAG-tagged Alex2 protein in the interstitial (Leydig) cell line TM3. A: Discreet pattern of Alex2 fusion protein expression throughout the cytoplasm of the cell (arrows). B: Distribution of endoplasmic reticulum (ER), as revealed by immunofluorescent staining for the ER-associated tetrapeptide KDEL (arrows). C: Distribution of the Golgi apparatus (arrows), as revealed by immunofluorescent detection of the Golgi-associated protein 58K. Nuclei stained blue with 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI).

Download figure to PowerPoint

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. REFERENCES

Whole-Mount In Situ Hybridization and Immunostaining

Whole mouse embryos or isolated urogenital systems (E9.5–E16.5) were fixed overnight at 4°C in 4% paraformaldehyde in phosphate buffered saline (PBS), and dehydrated in methanol. Whole-mount in situ hybridization using digoxigenin-labeled riboprobes was carried out as described previously (Andrews et al.,1998). An 800-bp sequence encompassing much of the mouse Alex2 unique region and 5′ untranslated region (UTR, Fig. 1) was used as a probe. PCR primers used to generate this probe were as follows: AlexA, 5′-GCC GTG TGG AAC TGT CTT TAT CCG-3′; and AlexB, 5′-TGG CGA AGG TAC CAC TGG CC-3′. The chromogen was 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT). Staining was only observed in tissues exposed to antisense probe; negative control sense probes produced no staining. For sectioning, some tissues were left in chromogen solution for an extended period (2 days), cryoprotected in 20% sucrose/PBS, embedded in OCT compound, and cut on a cryostat at 14 to 18 microns. Some sections were then immunostained using peroxidase-linked secondary antibodies or using immunofluorescence and Alexa-labeled secondary antibodies, as described previously (Smith et al.,2003).

RT-PCR

Total RNA was isolated from urogenital tissues or gonads and reverse-transcribed using oligo-dT and random hexamer primers, as described previously (Smith et al.,1999). For each sex at each day of development, several gonad pairs were pooled. Embryos were sexed by PCR amplification of Sry, with myogenin used as internal control, as described (McClive and Sinclair,2001). Specific PCR primers were designed to amplify a 380-bp fragment within the 5′-UTR of Alex2 cDNA. One primer spanned an exon–exon junction, precluding amplification of any contaminating genomic DNA. Thus, no Alex2 PCR products were seen in RT-minus samples (Fig. 3). Primers used for PCR were as follows: AlexC, 5′-TGT ACG GGC TTC ACA GGC TGG G-3′; and AlexD, 5′-GCA AGC TGA GCT GGA CCA GCT TCC-3′. PCR was carried out in the presence of 1.5 mM MgCl2, and the annealing temperature was 65°C. For semiquantitative RT-PCR, the linear range of amplification was first determined for Alex2 and Hprt. For both genes, 22 PCR cycles fell within this range and were chosen for routine analysis. Ethidium-stained bands were analyzed using the EagleEye II imaging system and EagleSight software (Stratagene). To demonstrate linearity, PCR reactions were also performed on serial dilutions of template DNA using 200 ng of E13.5 testis cDNA as starting template. For these experiments, linearity was demonstrated at both 22 and 30 PCR cycles. PCR products were resolved on a 1% agarose gel containing ethidium bromide. Bands were semiquantified using the integrated density function on ImageQuant software, as described previously (Smith et al.,1999).

Subcellular Localization

The open reading frame of mouse Alex2 was amplified from E12.5 mouse testis cDNA using Proofstart high-fidelity DNA polymerase (QIAGEN). The PCR product was cloned into pCMV-Tag4 expression vector (Stratagene) using restriction enzyme sites engineering into the primers. Using this system, Alex2 protein was tagged with FLAG epitope at the carboxy terminus. The insert was sequenced to confirm identity and purified plasmid transfected into TM4 or TM3 testicular cells (postnatal mouse Sertoli and Leydig cells, respectively). (RT-PCR analysis showed that both cell types express endogenous Alex2.) Lipofectamine 2000 was used for transfection. Twenty-four hours later, cells were fixed in cold methanol (10 min), followed by cold acetone (1 min) and incubated with rabbit-anti FLAG antibody, followed by Alexa 488-labeled goat anti-rabbit antibody (generating green immunofluorescence). In some cases, antibodies against endoplasmic reticulum (Stressgen) or Golgi apparatus (Sigma) were also used. Nuclei were counterstained with 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. REFERENCES