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- EXPERIMENTAL PROCEDURES
- Supporting Information
The tissues of the male reproductive tract are characterized by distinct morphologies, from highly coiled to un-coiled. Global gene expression profiles of efferent ducts, epididymis, and vas deferens were generated from embryonic day 14.5 to postnatal day 1 as tissue-specific morphologies emerge. Expression of homeobox genes, potential mediators of tissue-specific morphological development, was assessed. Twenty homeobox genes were identified as either tissue-enriched, developmentally regulated, or both. Additionally, ontology analysis demonstrated cell adhesion to be highly regulated along the length of the reproductive tract. Regulators of cell adhesion with variable expression between the three tissues were identified including Alcam, various cadherins, and multiple integrins. Immunofluorescence localization of the cell adhesion regulators POSTN and CDH2 demonstrated cell adhesion in the epithelium and mesenchyme of the epididymis may change throughout development. These results suggest cell adhesion may be modulated in a tissue-specific manner, playing an important role in establishing each tissue's final morphology. Developmental Dynamics 239:2479–2491, 2010. © 2010 Wiley-Liss, Inc.
- Top of page
- EXPERIMENTAL PROCEDURES
- Supporting Information
The efferent ducts, epididymis, and vas deferens comprise the reproductive excurrent duct system in the male. Each tissue is characterized by distinct morphologies and functions. There are three to five efferent ducts in the mouse, which are moderately coiled and give rise to a single common duct before transitioning into the epididymis (Joseph et al., 2009). One major role of the efferent ducts is the resorption of water (Clulow et al., 1994, 1998), under the control of both testosterone and estrogen (Hansen et al., 1997; Hess et al., 1997). The epididymis; a highly coiled, unbranched duct system is segmented into four gross anatomical regions, from cranial to caudal: the initial segment, caput, corpus, and cauda and has three major functions. Like the efferent ducts, the cranial portion of the epididymis concentrates spermatozoa. Additionally, the epididymis plays a key role in the maturation of spermatozoa to fully mature, motile sperm. Lastly, the caudal portion of the epididymis provides storage and protection for mature sperm before ejaculation. Distally, the epididymis terminates as the vas deferens. The vas deferens has the least complex ultrastructure, being uncoiled and unbranched, and serves primarily to store and protect mature sperm (Robaire and Hinton, 2002). During ejaculation, contractile waves in both the epididymis and vas deferens facilitate sperm expulsion from the excurrent ducts (Coolen et al., 2004; Vignozzi et al., 2008).
The majority of studies on the male excurrent duct system (reproductive tract) have focused on the expression and function of genes and proteins along the normal adult epididymal duct (Robaire and Hinton, 2002); however, proper development of all three tissues of the upper reproductive tract is essential for the maintenance of fertility. While there are very few genes that have been shown to be essential for male fertility in the adult epididymis, e.g., the forkhead box I1 (Foxi1) transcription factor (Blomqvist et al., 2006), there are several genes that likely play an important role during development of the epididymis, which in turn appear to be important for male fertility, e.g., Ros1 proto-oncogene (Ros1 or c-Ros; Sonnenberg-Riethmacher et al., 1996) and leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4; Mendive et al., 2006).
Although the final adult morphology and full cellular differentiation of the male reproductive tract is not complete until several weeks after birth (Robaire and Hinton, 2002), the period of late embryonic to early postnatal development is particularly important as it is during this time that the Wolffian duct undergoes regionalization and the individual tissues of the tract undergo the morphological changes that will eventually give rise to fully mature tissue (see Supp. Fig. S1, which is available online, for a brief overview and Joseph et al., 2009, for a detailed review). At embryonic day (E) 14.5, the upper reproductive tract consists of three distinct tube systems: the Wolffian duct, the mesonephric tubules, and the Mullerian duct. The upper portion of the Wolffian duct will give rise to the epididymis while the lower portion will form the vas deferens. The mesonephric tubules will eventually give rise to the efferent ducts. At E14.5, there is no morphological distinction between the upper and lower portion of the Wolffian duct, however by E16.5, the upper portion of the Wolffian duct has initiated the process of coiling while the Mullerian duct has fully regressed. During this period, the mesonephric tubules (putative efferent ducts) have also initiated coiling. From E16.5 to postnatal day (P) 1, coiling continues in the efferent ducts and moves caudally from the initial segment to the cauda of the developing epididymis. At no point in development does the vas deferens undergo coiling. Thus, regionalization of the Wolffian duct can be thought of as predominantly morphological with the upper portion diverging from the lower portion. However, increasing similarities in morphology are observed between the developing efferent ducts and epididymis, tissues derived from distinct embryonic structures.
Several factors have been shown to be important in the regionalization, elongation, or coiling of the developing reproductive tract. For example, animals lacking homeobox A10 (Hoxa10) or homeobox A11 (Hoxa11) have homeotic or partial homeotic transformations of the vas deferens to the epididymis (Hsieh-Li et al., 1995; Podlasek et al., 1999), linking the expression of Hox genes to regionalization of the Wolffian duct. Hox genes encode transcription factors that regulate anterior–posterior patterning and have been demonstrated to regulate the regionalization or patterning of multiple embryonic tube systems including the developing cardiac tube (Monier et al., 2007) and neural tube (Hidalgo-Sanchez et al., 2005; Ramos and Robert, 2005). Additionally, altered expression of Hox genes is known to have significant effects on the final morphology of moderately coiled tube systems, including the intestine (Wolgemuth et al., 1989; Zacchetti et al., 2007) and the kidney (Di-Poi et al., 2007). Unfortunately, a detailed analysis of Hox gene expression along the developing male reproductive tract has been unavailable.
Distinct morphologies are observed in the tissues of the male reproductive tract before and shortly after birth. To date, microarray analysis of this system has been limited to normal and treated adult epididymis (Wagenfeld et al., 2002; Ezer and Robaire, 2003; Xu et al., 2003; Chauvin and Griswold, 2004; Johnston et al., 2005; Yamazaki et al., 2006; Dube et al., 2007; Jelinsky et al., 2007; Turner et al., 2007) and treated adult efferent ducts (Snyder et al., 2009). There has been no global analysis examining the factors that govern the late embryonic and early postnatal development of the Wolffian and efferent ducts as well as those that regulate regionalization of the Wolffian duct. Tissue-specific global gene expression analysis throughout development provides a valuable resource for examining potential regulators of these processes. The global expression databases generated here will provide the opportunity for the research community to intensely study a dynamic period of development in three unique, but closely related reproductive tissues required for optimal fertility. Key factors involved in the development of these tissues are likely to serve homologous functions in other tissues and may provide insight into the mechanisms governing tubular and organ morphogenesis in the mammal.
- Top of page
- EXPERIMENTAL PROCEDURES
- Supporting Information
The Affymetrix microarray platform was used to determine global gene expression in three distinct tissues of the reproductive tract and their associated mesenchyme; the efferent ducts, the epididymis, and the vas deferens at four time points during development (E14.5, E16.5, E18.5, and P1). The resulting microarray output was used to determine transcripts that varied between tissues and throughout the developmental period. For the purpose of this work, the term tissue refers to both the ductal epithelium and the associated mesenchyme. Additionally, for clarity the terms efferent duct, epididymis, and vas deferens refer to the tissues as well as the structures from which they are derived. However, before E16.5 the epididymis and vas deferens combined would be more properly termed the Wolffian duct and before E18.5 the efferent ducts are more correctly called mesonephric tubules. Transcripts were considered enriched or reduced if the raw score was greater than 50 in all samples of the tissue/age of interest, the normalized value fold-change between the comparisons was greater than 2, and the comparison was found to be significant by two-way analysis of variance (ANOVA; P < 0.05). Selected comparisons were further screened by ontology analysis to determine common or enriched biological process, molecular functions, or cellular components (Dennis et al., 2003; Huang da et al., 2009). Microarray data was submitted to GEO under accession numbers GSM 560923 through 560946.
Validation of Tissue Collection and Array Output
To determine the accuracy and reproducibility of tissue collection, a heat map and hierarchical clustering analysis was generated using 18,845 probe IDs determined to be significantly different by two-way ANOVA (P < 0.05; Fig. 1). Clustering analysis revealed that duplicate samples for each time point were more similar to one another than other samples in all cases. Efferent duct samples were found to be more similar to one another than to all other tissues regardless of age, while early (E14.5 and E16.5) epididymis and vas deferens were more similar to each other than to late (E18.5 and P1) epididymis and vas deferens. Notably, late epididymis clustered more closely with efferent duct samples than with late vas deferens.
Figure 1. Hierarchical clustering of duplicate samples from the developing reproductive tract, embryonic day (E) 14.5 to postnatal day (P) 1.
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Isolation of the putative epididymis from the vas deferens at E14.5 is complicated by the lack of morphological markers to distinguish the two tissues while efferent ducts are easily isolated from the Wolffian duct based on tubule morphology (Supp. Fig. 2). As a result of this, isolation of epididymis from vas deferens was confirmed by expression analysis of known tissue-enriched genes (Fig. 2). Few tissue-enriched transcripts have been identified for the tissues derived from the Wolffian duct. To overcome this issue, a database of expression data for the urogenital tract, GUDMAP (www.gudmap.org) (Little et al., 2007) was queried to determine if transcripts identified as tissue-enriched at E14.5 for the epididymis and vas deferens had the expected message localization as demonstrated by in situ hybridization. Tissue-enrichment for decorin (Dcn; GUDMAP: 7307), integrin alpha 4 (Itga4; GUDMAP: 10897), and maternally expressed 3 (Meg3; GUDMAP: 9022) was observed in the putative epididymis while angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 (Ace2; GUDMAP: 9604), stanniocalcin 1 (Stc1; GUDMAP: 10864), and transmembrane protein 100 (Tmem100; GUDMAP: 7424) were observed in the putative vas deferens in this array analysis.
Figure 2. Expression profile of known tissue-enriched genes at embryonic day (E) 14.5 as determined by microarray. A: Transcripts enriched in the epididymis at E14.5. B: Transcripts enriched in the vas deferens at E14.5. Error bars represent standard deviation. Asterisks indicate significance (P > 0.05), determined by Tukey's HSD.
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Hox Gene Expression in the Developing Reproductive Tract
As key regulators of anterior–posterior patterning and tube regionalization, the expression profiles of Hox genes in the developing reproductive tract were examined. To define the appropriate data set for analysis, probe IDs of all genes with the word “homeobox” or “hox” in either the gene symbol, full name(s), or synonyms in the Gene Ontology (AmiGO) database were determined and used to query the reproductive tract developmental profile. The resulting probe ID list contained 254 probe IDs and targeted 172 Hox genes. Hox genes of interest included genes with tissue-enriched expression (Fig. 3) and those with unique expression profiles within a given tissue or set of tissues throughout development. Of the seven transcripts identified in this analysis as tissue-enriched throughout development (at least two-fold higher expression in one or more tissues as compared to the others at all time points and significantly greater at all time points as determined by two-way ANOVA with a P value < 0.05), two (Hoxd10 and Meis2) displayed a unique expression profile with increasing expression from the anterior (epididymis) to the posterior (vas deferens) of the Wolffian duct throughout development.
Figure 3. Tissue-enriched Hox transcripts in the developing reproductive tract. Dotted lines represent boundaries between tissues. ED (efferent ducts), Epi (epididymis), and VD (vas deferens).
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Developmental regulation of Hox gene expression is observed in many tissues, thus analysis of Hox genes with distinct expression profiles throughout development in one or more tissues of the reproductive tract was undertaken. Expression profiles were only considered of interest if the expression of a given gene varied at least two-fold throughout development in a given tissue and the change in expression throughout development was considered significant by two-way ANOVA (P < 0.05). Fifteen Hox genes were identified in this analysis (Supp. Fig. 3), one of which (Hoxd13) was also observed to have vas deferens-enriched expression. Eight Hox genes were observed to be developmentally regulated in only one of the three tissues: Dlx5, Hoxc10, and Hoxd13 in the vas deferens; Hoxa2, Hoxa4, Rhox2a, and Tlx2 in the efferent ducts; and Cux2 in the epididymis. The remaining six Hox genes were developmentally regulated in two or more of the tissues examined. Of these genes, developmental regulation between tissues was observed to be either similar or opposing in a gene-specific manner. Of particular interest were two genes with opposing developmental regulation (up in one tissue with age and down in another). These were Hoxd4 and Lhx1, both of which increased with age in the efferent ducts and decreased with age in the vas deferens.
Total Transcriptome Comparison of the Developing Efferent Ducts, Epididymis, and Vas Deferens
The total transcriptome for each tissue at each time point was determined by selecting for probe IDs having a raw score greater than 50 in both samples of a given tissue/age and being significantly different in at least one comparison across the entire experiment by two-way ANOVA (P < 0.05). Comparisons between tissues at each time point were then used to determine the percent similarity between the two transcriptomes, reported as % similar probes (Fig. 4). This analysis determined the epididymis and vas deferens transcriptomes were initially very similar but diverged (decreased in similarity) with age, the vas deferens and efferent ducts began and remained relatively dissimilar throughout the developmental period examined, and the epididymis and efferent ducts were relatively dissimilar at E14.5 but increased in similarity with age. As the total transcriptome comparisons model the changes in tissue morphology occurring within the tract, i.e., increasing similarity between the epididymis and efferent ducts (coiling) and decreasing similarity between the epididymis and the vas deferens (presence or absence of coiling) two comparisons were chosen to shed light on this phenomenon; epididymis-vs.-vas deferens and epididymis-vs.-efferent ducts.
Figure 4. Total transcriptome comparisons of efferent ducts, epididymis, and vas deferens throughout development. A: Epididymis vs. vas deferens. B: Vas deferens vs. efferent ducts. C: Epididymis vs. efferent ducts.
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Tissue to Tissue Comparisons Throughout Development
Epididymis and vas deferens comparisons.
Transcripts enriched in either the epididymis or vas deferens, as compared to one another, were identified and their associated biological functions, molecular processes, and cellular components determined by means of functional annotation clustering. Sixteen transcripts were enriched in the epididymis at all time points while 18 were enriched in the vas deferens at all time points. Functional annotation clustering demonstrated these genes were associated predominantly with three annotation clusters. The first included GO terms for regionalization and pattern specification process. Genes associated with this cluster consisted almost entirely of Hox genes. The second cluster included the GO term for tube morphogenesis and the last cluster consisted of GO terms for the cellular component extracellular matrix. The five transcripts with the highest tissue-enrichment throughout development when comparing epididymis to vas deferens are reported in Table 1.
Table 1. Genes Enriched in the Epididymis or Vas Deferens Throughout Development
| ||Genes enriched in the epididymis as compared to the vas deferens at all time points|| |
|Hoxc6||homeo box C6||NM_010465||6.6|
|Adamts16||a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 16||NM_172053||6.2|
|Lrrc17||leucine rich repeat containing 17||NM_028977||5.7|
|Ppp1r14a||protein phosphatase 1, regulatory (inhibitor) subunit 14A||NM_026731||5.1|
|Arhgap20||Rho GTPase activating protein 20||NM_175535||4.9|
| ||Genes enriched in the vas deferens as compared to the epididymis at all time points|| |
|Hoxd13||homeo box D13||NM_008275||24.5|
|Elavl2||ELAV (embryonic lethal, abnormal vision, Drosophila)-like 2 (Hu antigen B)||NM_207685||8.0|
|Kcnd2||potassium voltage-gated channel, Shal-related family, member 2||NM_019697||7.7|
|6330436F06Rik||RIKEN cDNA 6330436F06 gene|| ||7.6|
|Cxxc4||CXXC finger 4||NM_001004367||7.2|
Transcripts enriched in the epididymis or vas deferens at E14.5, the time before significant morphological changes in the tissue and thus potentially involved in early Wolffian duct regionalization, were identified. One hundred eighty-nine transcripts in the epididymis and 65 transcripts in the vas deferens were determined to be tissue-enriched at E14.5. Functional annotation clustering determined many of these genes were associated with the cellular component extracellular matrix and the biological processes tube development and skeletal system development. The top five tissue-enriched transcripts at E14.5 when comparing epididymis to vas deferens are reported in Table 2.
Table 2. Genes Enriched in the Epididymis or Vas Deferens at E14.5
|Genes enriched in the epididymis at E14.5 as compared to the vas deferens|
|Symbol||Name||RefSeq||Fold-change versus Vas deferens*|
|2810474O19Rik||RIKEN cDNA 2810474O19 gene|| ||9.6|
|Shisa3||shisa homolog 3 (Xenopus laevis)||NM_001033415||7.2|
|LOC100043998 /// Tpr||similar to nuclear pore complex-associated intranuclear coiled-coil protein TPR /// translocated promoter region|| ||5.6|
|Eif2c2||eukaryotic translation initiation factor 2C, 2||NM_153178||5.4|
|Itih5||inter-alpha (globulin) inhibitor H5||NM_172471||5|
|Genes enriched in the vas deferens at E14.5 as compared to the epididymis|
|Symbol||Name||RefSeq||Fold-change versus Epididymisa|
|Hoxd13||homeo box D13||NM_008275||10.4|
|Hoxa11||homeo box A11||NM_010450||8.2|
|Hoxd11||homeo box D11||NM_008273||6.7|
As significant morphological differentiation of the epididymis and the vas deferens occurs from E14.5 to after birth, genes associated with this process were of interest. When comparing epididymis to the vas deferens, genes associated with tissue-specific morphological changes would be expected to be similar in expression early in development but diverge in expression later. To that end, genes whose expression was similar in the two tissues (fold-change of < 2) at E14.5 but different (fold-change > 2) at later time points were identified. Of the genes with similar expression levels in the epididymis and vas deferens at E14.5; 75, 1770, and 2079 had higher transcript levels in the epididymis at E16.5, E18.5 and P1, respectively, and 79, 390, and 559 had higher transcript levels in the vas deferens at E16.5, E18.5, and P1, respectively. GO terms associated with cell adhesion were enriched in the epididymis at all time points and in the vas deferens at the later two time points. Terms associated with extracellular matrix were enriched in both tissues at E16.5 and in the vas deferens at E18.5. Notably, terms associated with apoptosis were enriched at E18.5 in the epididymis and at P1 in the vas deferens. GO terms associated with clusters having an enrichment score of greater than 2.0 in each tissue at each time point are reported in Supp. Table S1.
Epididymis and efferent ducts comparisons.
Transcripts enriched in either the epididymis or efferent ducts throughout development, as compared to one another, were identified and their associated biological functions, molecular processes, and cellular components determined by means of functional annotation clustering. Sixty-two transcripts were enriched in the epididymis at all time points while 26 were enriched in the efferent ducts. Functional annotation clustering demonstrated these genes were most commonly associated with the cellular component membrane and the biological processes of pattern specification and tube development. The five transcripts with the most enriched expression for each tissue throughout development are listed in Table 3.
Table 3. Genes Enriched in the Epididymis or Efferent Ducts Throughout Development
|Genes enriched in the epididymis as compared to the efferent ducts at all time points|
|Spink8||serine peptidase inhibitor, Kazal type 8||NM_183136||16.7|
|Hoxc10||homeo box C10||NM_010462||16.4|
|Rprm||reprimo, TP53 dependent G2 arrest mediator candidate||NM_023396||15.9|
|Hoxd10||homeo box D10||NM_013554||11.5|
|Genes enriched in the efferent ducts as compared to the epididymis at all time points|
|Pdzk1||PDZ domain containing 1||NM_021517||23.5|
|Keg1||kidney expressed gene 1||NM_029550||16.6|
|Aldh1l1||aldehyde dehydrogenase 1 family, member L1||NM_027406||8.7|
To identify genes associated with the increasing morphological similarity between the epididymis and efferent ducts, genes with different (> two-fold difference) expression in the epididymis and efferent ducts at E14.5 but similar (< two-fold difference) expression at later time points were identified. Of the genes expressed differently at E14.5; 430, 737, and 758 transcripts were expressed similarly at E16.5, E18.5, and P1, respectively, in the two tissues. These genes, like the genes identified in the epididymis and vas deferens comparisons, were commonly associated with the GO terms cell adhesion and extracellular matrix. Additionally, the genes expressed in a similar manner in the epididymis and efferent ducts with increasing age were also associated with the GO terms cell projection organization and cell motility. A summary of the functional annotation results for the epididymis and efferent ducts comparisons can be found in Supplemental Table S2.
Ontology Analysis Identification of Potential Morphological Regionalization Genes
To identify specific genes that may be playing a role in the morphological regionalization of the male reproductive tract, genes associated with the highly enriched GO term cell adhesion were identified and their tissue distribution and temporal expression patterns determined. This GO term was selected for further study for two reasons, it was identified as enriched throughout development in both tissue comparisons and regulation of cell adhesion is a potential mechanism for producing highly coiled tube structures within a mesenchymal tissue. In all, 92 individual genes were identified as associated with cell adhesion, 13 of which were identified in both the epididymis vs. vas deferens and epididymis vs. efferent ducts comparisons (Table 4). Of the 92 individual genes identified as associated with cell adhesion, potential players in establishing tissue-specific morphology were determined. A gene was considered a potential regulator of tissue-specific morphology if its expression change throughout development was found to be significant by two-way ANOVA (P < 0.05) and greater than two-fold in at least one tissue. Additionally, it was required to display either vas deferens enriched or epididymis and efferent duct enriched (significant by two-way ANOVA and two-fold or greater) expression at a minimum of one time point (Supp. Table S3). Of the genes passing criteria, 21 were vas deferens enriched and 9 epididymis and efferent duct enriched. Vas deferens enriched cell adhesion genes included three contactin genes, three genes encoding integrins, and five cadherin or protocadherin genes. Four well characterized cell adhesion genes were of particular interest: activated leukocyte cell adhesion molecule (Alcam), cadherin 2 (Cdh2), integrin alpha 1 (Itga1), and integrin alpha 8 (Itga8). The epididymis and efferent duct enriched genes included several additional genes of interest with clearly defined roles in cell adhesion: cysteine-rich protein 61 (Cyr61); integrin beta 1 binding protein 1 (Itgb1bp1); matrix metallopeptidase 14 (Mmp14); and periostin, osteoblast specific factor (Postn). Both Alcam and Postn expression can be observed histologically in the embryonic reproductive tract as early as E15.5 (GUDMAP: 11645 and 13280). The full expression profiles of all eight genes of interest can be found in Supplemental Figure 4.
Table 4. Cell Adhesion Genes Identified by Ontology Analysis of Both Tissue to Tissue Comparisons
|Bcl2||B-cell leukemia/lymphoma 2||NM_009741|
|Col8a1||collagen, type VIII, alpha 1||NM_007739|
|Cyr61||cysteine rich protein 61||NM_010516|
|Itga1||integrin alpha 1||NM_001033228|
|Itga4||integrin alpha 4||NM_010576|
|Lef1||lymphoid enhancer binding factor 1||NM_010703|
|Pard3||par-3 (partitioning defective 3) homolog (C. elegans)||NM_033620|
|Spp1||secreted phosphoprotein 1||NM_009263|
Periostin and cadherin 2 Protein Localization in the Developing Epididymis
The high expression of Postn in coiled tissues (the epididymis and efferent duct) relative to the vas deferens and its increasing expression with age suggested a potentially important role in reproductive tract tissue remodeling. To determine which cell populations were potentially undergoing POSTN mediated tissue remodeling, immunofluorescence was used to localize POSTN in the distal, developing epididymis (Fig. 5). Antibody specificity was confirmed by immunofluorescent detection of POSTN in POSTN positive control tissue (Supp. Fig. S5). At E14.5, POSTN is observed in both the epidermal and mesenchymal populations but is increasingly associated with the mesenchymal cell populations with age. By P1, POSTN is observed exclusively in the mesenchymal population of the epididymis. A notable shift from predominantly cytoplasmic to membrane or extracellular matrix associated is also observed with increasing age in the mesenchymal population as well (Supp. Fig. S5).
Figure 5. Immunofluorescent localization of POSTN in developing epididymis. A: Embryonic day (E) 14.5. B: E16.5. C: E18.5. D: P1 (inset: negative control). Mesenchymal cells marked by arrows and epididymal tubules denoted by T.
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The rapidly decreasing expression of Cdh2 in the epididymis suggested it may also be mediating tissue remodeling events. To determine the CDH2-positive cell populations in the epididymis and confirm a general reduction of CDH2 in the developing epididymis, immunofluorescence against CDH2 was performed on the distal, developing epididymis (Fig. 6). Throughout development, CDH2 is associated predominantly with the epithelial cells of the epididymis with some observable signal in the mesenchymal cells early in development, however by P1, CDH2 is observed exclusively in a small subpopulation of epithelial cells in the distal epididymis.
Figure 6. Immunofluorescent localization of CDH2 in developing epididymis. A: Embryonic day (E) 14.5. B: E16.5. C: E18.5. D: P1 (inset: negative control). Epithelial cells marked by open arrows and epididymal tubules denoted by T.
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