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Expression of the δ-protocadherin gene Pcdh19 in the developing mouse embryo
Gene Expression Patterns 6 (2006) 893–899 www.elsevier.com/locate/modgep
Expression of the d-protocadherin gene Pcdh19 in the developing mouse embryo Yaned Gaitan, Maxime Bouchard
*
McGill Cancer Centre and Biochemistry Department, McGill University, 3655 Promenade Sir-William-Osler, Montreal, Que., Canada H3G 1Y6 Received 12 January 2006; received in revised form 28 February 2006; accepted 1 March 2006 Available online 3 March 2006
Abstract Protocadherins constitute a large family of transmembrane proteins primarily involved in weak homophilic adhesion in the brain and several other tissues. In a screen for potential regulators of kidney development, we have identified Pcdh19, a poorly characterized member of the d-protocadherin subfamily. Here, we report the spatio-temporal expression pattern of Pcdh19 during mouse embryonic development. In midgestation embryos, Pcdh19 mRNA was detected in the mesonephros and in the neuroepithelium of the forebrain and midbrain. At later stages, Pcdh19 was expressed in other neural tissues such as the neural retina, nasal epithelium and spinal cord, as well as in the collecting duct and differentiating nephrons of the metanephros, in the glandular stomach, the exocrine pancreas and the hair follicles. Hence, the Pcdh19 gene is developmentally regulated during mouse organogenesis and shows a unique expression profile among protocadherins. Ó 2006 Elsevier B.V. All rights reserved. Keywords: d-Protocadherin; Pcdh19; Gene expression; Mouse; Embryo; Kidney; Brain; Spinal cord; Stomach; Neural retina; Nasal epithelium; Pancreas; Hair follicle
1. Results and discussion Protocadherins (Pcdh) form the largest of six (6) major cadherin subfamilies (Nollet et al., 2000). They are characterized by the presence of a large first exon coding for extracellular ectodomain repeats (typically six or seven), a transmembrane domain and for a part of the cytoplasmic domain (Frank and Kemler, 2002; Suzuki, 2000). At the cellular level, protocadherins are thought to act as weak homophilic adhesion molecules. Although most of the roughly 70 Pcdh genes are found in three clusters (Pcdh-a, Pcdh-b and Pcdh-c) located on chromosome 5q31, several others are found dispersed in the genome. Interestingly, 9 of the 14 non-clustered Pcdh gene products share a conserved cytoplasmic motif (CM2), which was first identified by homology between Pcdh8, Pcdh10, Pcdh18 and Pcdh19 (Frank and Kemler, 2002; Wolverton and Lalande, 2001). *
Corresponding author. Tel.: +1 514 398 3532; fax: +1 514 398 6769. E-mail address: [email protected] (M. Bouchard).
1567-133X/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2006.03.001
These proteins constitute the newly defined subfamily of d-protocadherins, further subdivided into d1- and d2protocadherins, based on the presence (d1) or absence (d2) of another cytoplasmic conserved motif (CM3) (Redies et al., 2005; Vanhalst et al., 2005). The CM3 motif was shown to specifically interact with protein phosphatase-1a (Vanhalst et al., 2005). Although the precise role of the CM2 domain remains unknown, the conservation of distinct cytoplasmic domains suggests that d-protocadherins share similar functional mechanisms. In contrast to the clustered Pcdh family members, which are mostly expressed in the central nervous system, d-protocadherins are additionally expressed in a variety of tissues such as the primitive streak, paraxial mesoderm, kidney, heart and lung (Hirano et al., 1999, 2001; Kuroda et al., 2002; Makarenkova et al., 2005; Murakami et al., 2005; Wolverton and Lalande, 2001; Yamamoto et al., 2000; Yoshida et al., 1999). Accordingly, functional studies reveal a role in somitogenesis, axial mesoderm patterning and gastrulation (Hukriede et al., 2003; Kuroda et al.,
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2002; Murakami et al., 2005). In addition, Pcdh10, a d2-protocadherin, was recently identified as a potential tumor suppressor gene in tumors derived from the nasopharynx, oesophagus and other tissues (Ying et al., 2006). Here, we report the expression pattern of the d2-protocadherin Pcdh19 in the developing mouse embryo. 1.1. Identification of Pcdh19 as a renal marker We used a BAC transgene expressing GFP under the control of the Pax2 locus (Pax2GFP) (Pfeffer et al., 2002) to specifically sort mesonephric cells from 9.5 days postcoitum (E9.5) mouse embryos. Total RNA from these cells was submitted to linear amplification and cDNA microarray analysis to determine the transcriptional profile of GFP(+) cells at different stages of development and in comparison to GFP() cells from the surrounding trunk (region between the hindbrain and tail bud, exclusively) (Grote et al., 2006). A selection for microarray signals strongly upregulated between 13-somite and 20-somite stages (E9.5) and specifically expressed in the mesonephros led to the identification of an expressed sequence tag (EST; BE652569) corresponding to the Pcdh19 gene (Unigene cluster Mm.39738; Table 1). The expression of Pcdh19 in human adult tissues was previously reported in the brain, heart, kidney, lung and trachea, using a RT-PCR approach (Wolverton and Lalande, 2001). 1.2. Early expression of pcdh19 To gain insight into the role of Pcdh19 in the developing embryo, we initially performed whole mount in situ hybridization at E9.0 and E10.5 with a specific cRNA probe. At E9.0, the expression of Pcdh19 was limited to a weak staining in the forebrain region (data not shown). By E9.5 the forebrain expression domain has become prominent, and extended to the entire midbrain as well as to a discrete domain of the hindbrain (Fig. 1A). At this stage, Pcdh19 expression was initiated in the presomitic mesoderm (Fig. 1A). These expression domains were maintained in E10.5 embryos, at which stage Pcdh19 expression also appeared in the otic vesicle, in the mesenchymal compartment of branchial arches 1 and 2, as well as in the foreand hindlimb bud mesenchyme (Fig. 1B, data not shown).
Table 1 Microarray analysis identifies Pcdh19 as a specific marker of the mesonephros
Pcdh19 b2-microglobulin
Mesonephros-specific expression
Upregulation 13s–16s
Upregulation 13s–20s
4.2 1.0
2.3 1.0
2.8 1.2
Ratios are expressed as GFP+/GFP or late/early. Embryonic stages are defined in somite number (s) of E9.0 to E9.5 embryos. b2-microglobulin serves as a housekeeping gene control.
We next performed in situ hybridization analysis on sagittal sections of E12.5 embryos. At that stage, the brain has undergone extensive morphogenic changes generating complex neural structures that are undergoing cellular differentiation. Pcdh19 mRNA was detected at low level in most of the neuroepithelium of the brain with discrete regions of high expression, notably in the roof of the neopallial cortex and in the diencephalon (Fig. 1C). At E12.5, Pcdh19 expression was also detected in the spinal cord and in internal tissues such as the stomach and dorsal mesentery (Fig. 1C). 1.3. Expression of Pcdh19 in the head and spinal cord As the early expression of Pcdh19 is predominantly neural, we proceeded to characterize the head and spinal cord expression patterns in more details. At E12.5, transversal sections in the head region revealed graded expression of Pcdh19 along the dorsal cortex and in the lateral ganglionic eminence (Fig. 2A). Pcdh19 transcript was also detected in the neural retina and in the epithelium lining the nasal cavity of E12.5 embryos (Fig. 2A), and remained present at E15.5 (Figs. 2B and D). Sections in the head region further identified mesenchymal domains (Figs. 2A and B) as well as the Rathke’s pouch, primordium of the pituitary gland, to be positive for Pcdh19. In the spinal cord, Pcdh19 mRNA was detected exclusively in post-mitotic neurons, with the highest levels found in a population of Pax2-positive ventral interneurons (Figs. 2E and F). Strong spinal cord expression was maintained in E15.5 and E18.5 embryos (Figs. 2G and H). Hence, as most other protocadherins, Pcdh19 shows an elaborate expression profile in neural tissues. 1.4. Expression of Pcdh19 in the kidney The initial microarray identification of Pcdh19 as a renal marker prompted us to perform a detailed characterization at different stages of kidney development. In the embryo, kidney development is initiated by the formation of the pro/mesonephros, that is generated by the caudal elongation of the nephric duct, which induces mesonephric tubule formation in the adjacent nephric cord (Bouchard, 2004). The metanephros, or adult kidney, forms in the caudal region of the nephric duct and is characterized by iterative branching of the collecting system, the tips of which induce nephron formation through a series of well-defined stages (Saxen, 1987). In agreement with its initial identification by microarray analysis, Pcdh19 transcription was detected in the nephric duct and nephric cord of E9.5 mesonephros (Fig. 3A). By E10.5, expression was maintained in the nephric duct and additionally found in the mesonephric tubules. In these tissues, Pcdh19 was coexpressed with the kidney developmental regulator Pax2 (Fig. 3B) (Bouchard et al., 2002; Torres et al., 1995). However, in situ analysis in embryos mutant for Pax2 and its paralog Pax8 (Pax2/Pax8+/) did not
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Fig. 1. Early expression of Pcdh19. In situ hybridization for Pcdh19 on whole mount (A and B) and sagittal cryosection (C). (A) At E9.5, Pcdh19 transcript is detected in the brain and presomitic mesoderm. (B) At E10.5, brain expression is maintained and additional expression is detected in the brachial arches, otic vesicles, limb buds and caudal nephric duct. (C) In E12.5 embryos, high mRNA levels were observed in specific regions of the brain, in the epithelium lining the nasal cavity, spinal cord, stomach region and dorsal mesenteries. ba, branchial arch; di, diencephalons; dmes, dorsal mesentery; ep, epiphysis; fb, forebrain; hb, hindbrain; lb, limb bud; mb, midbrain; nd, nephric duct; oe, olfactory epithelium; ov, otic vesicle; psm, presomitic mesoderm; sc, spinal cord; st, stomach. Scale bars, 500 lm.
result in a significant reduction in Pcdh19 expression levels, arguing against a genetic regulation of Pcdh19 by Pax proteins (data not shown). At E12.5, when metanephros development is well initiated, Pcdh19 was detected in the ureter tip and collecting duct as well as in the newly epithelialized renal vesicles (Fig. 3C). It was, however, absent from the condensing mesenchyme from which the vesicles are derived (Fig. 3C). Similar results were obtained in E15.5 and E18.5 metanephros, in which additional expression was detected in comma- and S-shaped bodies as well as in the tubular nephron (Fig. 3D and data not shown). Together, these results identify Pcdh19 as an epithelial marker of the embryonic kidney. 1.5. Expression of Pcdh19 in other tissues In addition to the CNS and kidney, Pcdh19 transcriptional activity could be detected in several other tissues during embryonic development. In E12.5 embryos, Pcdh19 was found in the dermomyotome (Fig. 4A), as well as in the dorsal and urogenital mesenteries, which will later form
connective tissues (Fig. 4B). At this stage, Pcdh19 transcript was also present in the mesenchyme surrounding the forestomach, but absent from the hindstomach mesenchyme. Such restricted gene expression domains along the stomach primordium is observed for several other developmental genes such as Shh and BMP4, and reflects the active patterning occurring at this stage (Bitgood and McMahon, 1995). Interestingly, Pcdh19 formed a ring of expression around the endodermal tissue that remains closed at this stage, while the mesenchyme surrounding the expanding endoderm was devoid of Pcdh19 mRNA. At later stages of stomach development, Pcdh19 was detected in the gland epithelium and mesenchyme of the gastric units but absent from the luminal epithelium (Figs. 4D and E). In the digestive system, Pcdh19 was additionally found at high levels in the pancreas and duodenum (Fig. 4F and data not shown). Another prominent expression domain of Pcdh19 is the hair follicle. In the mouse, this mini-organ is induced by mesenchymal–epithelial interaction at E14.5–E15.5 and progresses through several morphogenetic stages to reach maturity by E18.5 (Schmidt-Ullrich and Paus, 2005). At
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Fig. 2. Expression of Pcdh19 in neural tissues. In situ hybridization for Pcdh19 on longitudinal (A), sagittal (C) and transversal (B, D and E–H) sections at E12.5 (A, C, E and F), E15.5 (B, D and G) and E18.5 (H). (A) Expression is detected in the cortex, lateral ganglionic eminence, neural retina and nasal epithelium. (B) High mRNA levels are observed in the epithelium lining the nasal cavity. (C) Expression in Rathke’s pouch and diencephalon. (D) The neural retina and primary lens fiber cells express high levels of Pcdh19. (E) In the spinal cord, expression is mostly detected in post-mitotic interneurons. (F) Spinal cord signal overlaps with Pax2 (brown) in a subset of ventral interneurons (arrowhead). (G and H) High mRNA levels are maintained in the spinal cord at E15.5 and E18.5. cor, cortex; di, diencephalon; drg, dorsal root ganglion; enc, epithelial lining of nasal cavity; in, interneurons; lf, primary lens fibers; lge, lateral ganglionic eminence; m, mesenchyme; nr, neural retina; rp, Rathke’s pouch. Scale bars: (A) 400 lm, (B–H) 200 lm.
E15.5, Pcdh19 expression could already be detected in the mesenchymal component of the hair follicle primordium (Fig. 4G). In more mature follicles, Pcdh19 transcription
was present both in the dermal papilla and matrix (Fig. 4H). A similar expression profile was observed in the differentiating whiskers between E12.5 and E15.5 (data
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Fig. 3. Expression of Pcdh19 in the renal system. (A) At E9.5, specific expression is detected in the nephric duct and nephric cord of the mesonephros. (B) At E10.5, Pcdh19 expression (blue) coincides with Pax2 (red-brown) in the nephric duct and mesonephric tubules. (C) Counterstaining with Pax2 (redbrown) reveals the presence of Pcdh19 (blue) in the ureter tip, collecting duct and renal vesicles of the early metanephros but absence of expression in the condensing mesenchyme at E12.5. Low levels were observed in the mesenchyme surrounding the kidney. (D) At E18.5, Pcdh19 is maintained in the ureter tip and renal vesicle and is now detectable in the comma-shaped bodies and at low levels in distal convoluted tubules. Glomeruli are negative for Pcdh19 expression. cd, collecting duct; cm, condensing mesenchyme; cb, comma-shaped body; cd, collecting duct; cm, condensing mesenchyme; dct, distal convoluted tubule; g, glomerulus; mst, mesonephric tubule; nc, nephric cord; nd, nephric duct; rv, renal vesicle; sb, s-shaped body; sm, surrounding mesenchyme; ut, ureter tip. Scale bars, 50 lm.
not shown). These analyses further revealed a diffuse expression of Pcdh19 on the mesenchymal layers beneath the skin (Fig. 4G). Altogether, these data clearly demonstrate the fine regulation of Pcdh19 in specific cell types of several tissues undergoing active morphogenesis.
et al., 2000) and the Xenopus Pcdh1, found in the brain, kidney, somite and otic vesicle (Kuroda et al., 2002). However, the expression profile described here is unique among the protocadherins documented to date. Hence, these data constitute a strong basis for elucidating the function of Pcdh19 in the developing embryo.
1.6. Conclusions In a search for potential developmental regulators of kidney development, we have identified Pcdh19, a poorly characterized member of the protocadherin family. Using in situ hybridization, we showed that Pcdh19 is expressed in a tissue-specific manner during embryogenesis. Pcdh19 was found in the epithelial compartments of the kidney, olfactory system, eye, brain and late stomach and in the mesenchymal compartment of the early stomach, dermomyotome, mesenteries, dermal papilla and presomitic mesoderm. Interestingly, in most of these tissues Pcdh19 expression is first detected at the early stages of organ patterning. The tissue distribution during embryogenesis shows some overlap with other members of the d-protocadherin family, notably with mouse and zebrafish Pcdh8 and Pcdh10, which are also expressed in the brain, olfactory epithelium and paraxial mesoderm (Hirano et al., 1999; Makarenkova et al., 2005; Murakami et al., 2005; Yamamoto
2. Experimental procedures 2.1. Mice Pax2GFP BAC transgene (#30) used to sort mesonephros-specific cells was described previously (Grote et al., 2006; Pfeffer et al., 2002). Embryos for gene expression analysis were generated by timed matings of wild-type CD-1 mice. Noon of the day of plug detection was considered as E0.5.
2.2. Microarray analysis FACS sorting of Pax2(+) mesonephric cells, linear amplification and DNA microarray analysis were described previously (Bouchard et al., 2005; Grote et al., 2006).
2.3. In situ hybridization and immunohistochemistry Dissected embryos were fixed at 4 °C for 4 h to overnight in 4% paraformaldehyde and dehydrated in methanol series for whole mount in situ hybridization or equilibrated in 30% sucrose and mounted in OCT (Tissue-Tek) for cryosectioning. In situ hybridizations on whole
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Fig. 4. Expression of Pcdh19 in other tissues. In situ hybridization for Pcdh19 on transversal sections at E12.5 (A–C), E15.5 (G) and E18.5 (D–F and H). (A) Transcription signal is detected in the dermomyotome and spinal cord. (B) Urogenital and dorsal mesenteries show diffuse mRNA signal at the level of the mesonephros. (C) Pcdh19 is detected specifically in the mesenchyme surrounding the forestomach endoderm. (D) At E18.5, staining is observed in the glandular stomach. (E) At higher magnification, the expression can be found in the glandular epithelium and mesenchyme while the luminal epithelium is negative for Pcdh19. (F) Expression in the exocrine pancreas and oesophagus. (G) Onset of Pcdh19 expression in the hair follicle primordium. (H) Strong signal is observed in the dermal papilla and matrix of the mature hair follicle. dp, dermal papilla; dm, dermomyotome; dmes, dorsal mesentery; fsm, forestomach mesenchyme; ge, glandular epithelium; gm, glandular mesenchyme; gst, glandular stomach; hfp, hair follicle primordium; hsm, hindstomach mesenchyme; le, luminal epithelium; m, mesenchyme; ma, matrix; mn, mesonephros; oes, oesophagus; pa, pancreas; sc, spinal cord; umes, urogenital mesentery. Scale bars: (A and F) 200 lm, (B–D) 100 lm, (E, G and H) 50 lm. mount and sections were performed as described (Henrique et al., 1995; Hynes et al., 2000). Pcdh19 cRNA probe was generated by RT-PCR from E17.5 kidney cDNA using the following primers: 5 0 -CACCAAGCAGAA GATTGACCGAG-3 0 and 5 0 -GCCTCCCATCCACAAGAATAGTG-3 0 .
The 986 bp PCR product, corresponding to positions 1963–2949 of the reference sequence XM_205287 (exon 1), was cloned in pGEM-T-easy and used for digoxigenin-dUTP cRNA probe synthesis, following linearization. For double stainings, in situ hybridization was performed first,
Y. Gaitan, M. Bouchard / Gene Expression Patterns 6 (2006) 893–899 followed by immunohistochemistry as described previously (Bouchard et al., 2000). Immunodetection of the rabbit anti-Pax2 antibody (1:100 dilution, Covance) was revealed with the ABC Vectastain kits (Vector labs).
Acknowledgements We thank Susanne Kaitna and the members of the Bouchard’s laboratory for critical reading of the manuscript and Fre´deric Charron for expertise on neural expression. This work was supported by the Canadian Institutes for Health Research and The Terry Fox Foundation. M.B holds a Canada Research Chair in Kidney Disease. References Bitgood, M.J., McMahon, A.P., 1995. Hedgehog and Bmp genes are coexpressed at many diverse sites of cell–cell interaction in the mouse embryo. Dev. Biol. 172, 126–138. Bouchard, M., 2004. Transcriptional control of kidney development. Differentiation 72, 295–306. Bouchard, M., Grote, D., Craven, S.E., Sun, Q., Steinlein, P., Busslinger, M., 2005. Identification of Pax2-regulated genes by expression profiling of the mid-hindbrain organizer region. Development 132, 2633–2643. Bouchard, M., Pfeffer, P., Busslinger, M., 2000. Functional equivalence of the transcription factors Pax2 and Pax5 in mouse development. Development 127, 3703–3713. Bouchard, M., Souabni, A., Mandler, M., Neubuser, A., Busslinger, M., 2002. Nephric lineage specification by Pax2 and Pax8. Genes Dev. 16, 2958–2970. Frank, M., Kemler, R., 2002. Protocadherins. Curr. Opin. Cell Biol. 14, 557–562. Grote, D., Souabni, A., Busslinger, M., Bouchard, M., 2006. Pax2/8regulated Gata3 expression is necessary for morphogenesis and guidance of the nephric duct in the developing kidney. Development 133, 53–61. Henrique, D., Adam, J., Myat, A., Chitnis, A., Lewis, J., Ish-Horowicz, D., 1995. Expression of a Delta homologue in prospective neurons in the chick. Nature 375, 787–790. Hirano, S., Yan, Q., Suzuki, S.T., 1999. Expression of a novel protocadherin, OL-protocadherin, in a subset of functional systems of the developing mouse brain. J. Neurosci. 19, 995–1005. Homayouni, R., Rice, D.S., Curran, T., 2001. Disabled-1 interacts with a novel developmentally regulated protocadherin. Biochem. Biophys. Res. Commun. 289, 539–547. Hukriede, N.A., Tsang, T.E., Habas, R., Khoo, P.L., Steiner, K., Weeks, D.L., Tam, P.P., Dawid, I.B., 2003. Conserved requirement of Lim1 function for cell movements during gastrulation. Dev. Cell 4, 83–94.
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Hynes, M., Ye, W., Wang, K., Stone, D., Murone, M., Sauvage, F., Rosenthal, A., 2000. The seven-transmembrane receptor smoothened cell-autonomously induces multiple ventral cell types. Nat. Neurosci. 3, 41–46. Kuroda, H., Inui, M., Sugimoto, K., Hayata, T., Asashima, M., 2002. Axial protocadherin is a mediator of prenotochord cell sorting in Xenopus. Dev. Biol. 244, 267–277. Makarenkova, H., Sugiura, H., Yamagata, K., Owens, G., 2005. Alternatively spliced variants of protocadherin 8 exhibit distinct patterns of expression during mouse development. Biochim. Biophys. Acta 1681, 150–156. Murakami, T., Hijikata, T., Matsukawa, M., Ishikawa, H., Yorifuji, H., 2005. Zebrafish protocadherin 10 is involved in paraxial mesoderm development and somitogenesis. Dev. Dyn. 235, 506–514. Nollet, F., Kools, P., van Roy, F., 2000. Phylogenetic analysis of the cadherin superfamily allows identification of six major subfamilies besides several solitary members. J. Mol. Biol. 299, 551–572. Pfeffer, P.L., Payer, B., Reim, G., di Magliano, M.P., Busslinger, M., 2002. The activation and maintenance of Pax2 expression at the midhindbrain boundary is controlled by separate enhancers. Development 129, 307–318. Redies, C., Vanhalst, K., Roy, F., 2005. delta-Protocadherins: unique structures and functions. Cell Mol. Life Sci. 62, 2840–2852. Saxen, L. (Ed.), 1987. Organogenesis of the kidney. Developmental and Cell Biology Series, vol. 19. Cambridge University Press, Cambridge, UK, p. 165. Schmidt-Ullrich, R., Paus, R., 2005. Molecular principles of hair follicle induction and morphogenesis. Bioessays 27, 247–261. Suzuki, S.T., 2000. Recent progress in protocadherin research. Exp. Cell Res. 261, 13–18. Torres, M., Gomez-Pardo, E., Dressler, G.R., Gruss, P., 1995. Pax-2 controls multiple steps of urogenital development. Development 121, 4057–4065. Vanhalst, K., Kools, P., Staes, K., van Roy, F., Redies, C., 2005. deltaProtocadherins: a gene family expressed differentially in the mouse brain. Cell Mol. Life Sci. 62, 1247–1259. Wolverton, T., Lalande, M., 2001. Identification and characterization of three members of a novel subclass of protocadherins. Genomics 76, 66–72. Yamamoto, A., Kemp, C., Bachiller, D., Geissert, D., De Robertis, E.M., 2000. Mouse paraxial protocadherin is expressed in trunk mesoderm and is not essential for mouse development. Genesis 27, 49–57. Ying, J., Li, H., Seng, T.J., Langford, C., Srivastava, G., Tsao, S.W., Putti, T., Murray, P., Chan, A.T., Tao, Q., 2006. Functional epigenetics identifies a protocadherin PCDH10 as a candidate tumor suppressor for nasopharyngeal, esophageal and multiple other carcinomas with frequent methylation. Oncogene 25, 1070–1080. Yoshida, K., Watanabe, M., Kato, H., Dutta, A., Sugano, S., 1999. BHprotocadherin-c, a member of the cadherin superfamily, interacts with protein phosphatase 1 alpha through its intracellular domain. FEBS Lett. 460, 93–98.
Expression of the d-protocadherin gene Pcdh19 in the developing mouse embryo Yaned Gaitan, Maxime Bouchard
*
McGill Cancer Centre and Biochemistry Department, McGill University, 3655 Promenade Sir-William-Osler, Montreal, Que., Canada H3G 1Y6 Received 12 January 2006; received in revised form 28 February 2006; accepted 1 March 2006 Available online 3 March 2006
Abstract Protocadherins constitute a large family of transmembrane proteins primarily involved in weak homophilic adhesion in the brain and several other tissues. In a screen for potential regulators of kidney development, we have identified Pcdh19, a poorly characterized member of the d-protocadherin subfamily. Here, we report the spatio-temporal expression pattern of Pcdh19 during mouse embryonic development. In midgestation embryos, Pcdh19 mRNA was detected in the mesonephros and in the neuroepithelium of the forebrain and midbrain. At later stages, Pcdh19 was expressed in other neural tissues such as the neural retina, nasal epithelium and spinal cord, as well as in the collecting duct and differentiating nephrons of the metanephros, in the glandular stomach, the exocrine pancreas and the hair follicles. Hence, the Pcdh19 gene is developmentally regulated during mouse organogenesis and shows a unique expression profile among protocadherins. Ó 2006 Elsevier B.V. All rights reserved. Keywords: d-Protocadherin; Pcdh19; Gene expression; Mouse; Embryo; Kidney; Brain; Spinal cord; Stomach; Neural retina; Nasal epithelium; Pancreas; Hair follicle
1. Results and discussion Protocadherins (Pcdh) form the largest of six (6) major cadherin subfamilies (Nollet et al., 2000). They are characterized by the presence of a large first exon coding for extracellular ectodomain repeats (typically six or seven), a transmembrane domain and for a part of the cytoplasmic domain (Frank and Kemler, 2002; Suzuki, 2000). At the cellular level, protocadherins are thought to act as weak homophilic adhesion molecules. Although most of the roughly 70 Pcdh genes are found in three clusters (Pcdh-a, Pcdh-b and Pcdh-c) located on chromosome 5q31, several others are found dispersed in the genome. Interestingly, 9 of the 14 non-clustered Pcdh gene products share a conserved cytoplasmic motif (CM2), which was first identified by homology between Pcdh8, Pcdh10, Pcdh18 and Pcdh19 (Frank and Kemler, 2002; Wolverton and Lalande, 2001). *
Corresponding author. Tel.: +1 514 398 3532; fax: +1 514 398 6769. E-mail address: [email protected] (M. Bouchard).
1567-133X/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2006.03.001
These proteins constitute the newly defined subfamily of d-protocadherins, further subdivided into d1- and d2protocadherins, based on the presence (d1) or absence (d2) of another cytoplasmic conserved motif (CM3) (Redies et al., 2005; Vanhalst et al., 2005). The CM3 motif was shown to specifically interact with protein phosphatase-1a (Vanhalst et al., 2005). Although the precise role of the CM2 domain remains unknown, the conservation of distinct cytoplasmic domains suggests that d-protocadherins share similar functional mechanisms. In contrast to the clustered Pcdh family members, which are mostly expressed in the central nervous system, d-protocadherins are additionally expressed in a variety of tissues such as the primitive streak, paraxial mesoderm, kidney, heart and lung (Hirano et al., 1999, 2001; Kuroda et al., 2002; Makarenkova et al., 2005; Murakami et al., 2005; Wolverton and Lalande, 2001; Yamamoto et al., 2000; Yoshida et al., 1999). Accordingly, functional studies reveal a role in somitogenesis, axial mesoderm patterning and gastrulation (Hukriede et al., 2003; Kuroda et al.,
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2002; Murakami et al., 2005). In addition, Pcdh10, a d2-protocadherin, was recently identified as a potential tumor suppressor gene in tumors derived from the nasopharynx, oesophagus and other tissues (Ying et al., 2006). Here, we report the expression pattern of the d2-protocadherin Pcdh19 in the developing mouse embryo. 1.1. Identification of Pcdh19 as a renal marker We used a BAC transgene expressing GFP under the control of the Pax2 locus (Pax2GFP) (Pfeffer et al., 2002) to specifically sort mesonephric cells from 9.5 days postcoitum (E9.5) mouse embryos. Total RNA from these cells was submitted to linear amplification and cDNA microarray analysis to determine the transcriptional profile of GFP(+) cells at different stages of development and in comparison to GFP() cells from the surrounding trunk (region between the hindbrain and tail bud, exclusively) (Grote et al., 2006). A selection for microarray signals strongly upregulated between 13-somite and 20-somite stages (E9.5) and specifically expressed in the mesonephros led to the identification of an expressed sequence tag (EST; BE652569) corresponding to the Pcdh19 gene (Unigene cluster Mm.39738; Table 1). The expression of Pcdh19 in human adult tissues was previously reported in the brain, heart, kidney, lung and trachea, using a RT-PCR approach (Wolverton and Lalande, 2001). 1.2. Early expression of pcdh19 To gain insight into the role of Pcdh19 in the developing embryo, we initially performed whole mount in situ hybridization at E9.0 and E10.5 with a specific cRNA probe. At E9.0, the expression of Pcdh19 was limited to a weak staining in the forebrain region (data not shown). By E9.5 the forebrain expression domain has become prominent, and extended to the entire midbrain as well as to a discrete domain of the hindbrain (Fig. 1A). At this stage, Pcdh19 expression was initiated in the presomitic mesoderm (Fig. 1A). These expression domains were maintained in E10.5 embryos, at which stage Pcdh19 expression also appeared in the otic vesicle, in the mesenchymal compartment of branchial arches 1 and 2, as well as in the foreand hindlimb bud mesenchyme (Fig. 1B, data not shown).
Table 1 Microarray analysis identifies Pcdh19 as a specific marker of the mesonephros
Pcdh19 b2-microglobulin
Mesonephros-specific expression
Upregulation 13s–16s
Upregulation 13s–20s
4.2 1.0
2.3 1.0
2.8 1.2
Ratios are expressed as GFP+/GFP or late/early. Embryonic stages are defined in somite number (s) of E9.0 to E9.5 embryos. b2-microglobulin serves as a housekeeping gene control.
We next performed in situ hybridization analysis on sagittal sections of E12.5 embryos. At that stage, the brain has undergone extensive morphogenic changes generating complex neural structures that are undergoing cellular differentiation. Pcdh19 mRNA was detected at low level in most of the neuroepithelium of the brain with discrete regions of high expression, notably in the roof of the neopallial cortex and in the diencephalon (Fig. 1C). At E12.5, Pcdh19 expression was also detected in the spinal cord and in internal tissues such as the stomach and dorsal mesentery (Fig. 1C). 1.3. Expression of Pcdh19 in the head and spinal cord As the early expression of Pcdh19 is predominantly neural, we proceeded to characterize the head and spinal cord expression patterns in more details. At E12.5, transversal sections in the head region revealed graded expression of Pcdh19 along the dorsal cortex and in the lateral ganglionic eminence (Fig. 2A). Pcdh19 transcript was also detected in the neural retina and in the epithelium lining the nasal cavity of E12.5 embryos (Fig. 2A), and remained present at E15.5 (Figs. 2B and D). Sections in the head region further identified mesenchymal domains (Figs. 2A and B) as well as the Rathke’s pouch, primordium of the pituitary gland, to be positive for Pcdh19. In the spinal cord, Pcdh19 mRNA was detected exclusively in post-mitotic neurons, with the highest levels found in a population of Pax2-positive ventral interneurons (Figs. 2E and F). Strong spinal cord expression was maintained in E15.5 and E18.5 embryos (Figs. 2G and H). Hence, as most other protocadherins, Pcdh19 shows an elaborate expression profile in neural tissues. 1.4. Expression of Pcdh19 in the kidney The initial microarray identification of Pcdh19 as a renal marker prompted us to perform a detailed characterization at different stages of kidney development. In the embryo, kidney development is initiated by the formation of the pro/mesonephros, that is generated by the caudal elongation of the nephric duct, which induces mesonephric tubule formation in the adjacent nephric cord (Bouchard, 2004). The metanephros, or adult kidney, forms in the caudal region of the nephric duct and is characterized by iterative branching of the collecting system, the tips of which induce nephron formation through a series of well-defined stages (Saxen, 1987). In agreement with its initial identification by microarray analysis, Pcdh19 transcription was detected in the nephric duct and nephric cord of E9.5 mesonephros (Fig. 3A). By E10.5, expression was maintained in the nephric duct and additionally found in the mesonephric tubules. In these tissues, Pcdh19 was coexpressed with the kidney developmental regulator Pax2 (Fig. 3B) (Bouchard et al., 2002; Torres et al., 1995). However, in situ analysis in embryos mutant for Pax2 and its paralog Pax8 (Pax2/Pax8+/) did not
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Fig. 1. Early expression of Pcdh19. In situ hybridization for Pcdh19 on whole mount (A and B) and sagittal cryosection (C). (A) At E9.5, Pcdh19 transcript is detected in the brain and presomitic mesoderm. (B) At E10.5, brain expression is maintained and additional expression is detected in the brachial arches, otic vesicles, limb buds and caudal nephric duct. (C) In E12.5 embryos, high mRNA levels were observed in specific regions of the brain, in the epithelium lining the nasal cavity, spinal cord, stomach region and dorsal mesenteries. ba, branchial arch; di, diencephalons; dmes, dorsal mesentery; ep, epiphysis; fb, forebrain; hb, hindbrain; lb, limb bud; mb, midbrain; nd, nephric duct; oe, olfactory epithelium; ov, otic vesicle; psm, presomitic mesoderm; sc, spinal cord; st, stomach. Scale bars, 500 lm.
result in a significant reduction in Pcdh19 expression levels, arguing against a genetic regulation of Pcdh19 by Pax proteins (data not shown). At E12.5, when metanephros development is well initiated, Pcdh19 was detected in the ureter tip and collecting duct as well as in the newly epithelialized renal vesicles (Fig. 3C). It was, however, absent from the condensing mesenchyme from which the vesicles are derived (Fig. 3C). Similar results were obtained in E15.5 and E18.5 metanephros, in which additional expression was detected in comma- and S-shaped bodies as well as in the tubular nephron (Fig. 3D and data not shown). Together, these results identify Pcdh19 as an epithelial marker of the embryonic kidney. 1.5. Expression of Pcdh19 in other tissues In addition to the CNS and kidney, Pcdh19 transcriptional activity could be detected in several other tissues during embryonic development. In E12.5 embryos, Pcdh19 was found in the dermomyotome (Fig. 4A), as well as in the dorsal and urogenital mesenteries, which will later form
connective tissues (Fig. 4B). At this stage, Pcdh19 transcript was also present in the mesenchyme surrounding the forestomach, but absent from the hindstomach mesenchyme. Such restricted gene expression domains along the stomach primordium is observed for several other developmental genes such as Shh and BMP4, and reflects the active patterning occurring at this stage (Bitgood and McMahon, 1995). Interestingly, Pcdh19 formed a ring of expression around the endodermal tissue that remains closed at this stage, while the mesenchyme surrounding the expanding endoderm was devoid of Pcdh19 mRNA. At later stages of stomach development, Pcdh19 was detected in the gland epithelium and mesenchyme of the gastric units but absent from the luminal epithelium (Figs. 4D and E). In the digestive system, Pcdh19 was additionally found at high levels in the pancreas and duodenum (Fig. 4F and data not shown). Another prominent expression domain of Pcdh19 is the hair follicle. In the mouse, this mini-organ is induced by mesenchymal–epithelial interaction at E14.5–E15.5 and progresses through several morphogenetic stages to reach maturity by E18.5 (Schmidt-Ullrich and Paus, 2005). At
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Fig. 2. Expression of Pcdh19 in neural tissues. In situ hybridization for Pcdh19 on longitudinal (A), sagittal (C) and transversal (B, D and E–H) sections at E12.5 (A, C, E and F), E15.5 (B, D and G) and E18.5 (H). (A) Expression is detected in the cortex, lateral ganglionic eminence, neural retina and nasal epithelium. (B) High mRNA levels are observed in the epithelium lining the nasal cavity. (C) Expression in Rathke’s pouch and diencephalon. (D) The neural retina and primary lens fiber cells express high levels of Pcdh19. (E) In the spinal cord, expression is mostly detected in post-mitotic interneurons. (F) Spinal cord signal overlaps with Pax2 (brown) in a subset of ventral interneurons (arrowhead). (G and H) High mRNA levels are maintained in the spinal cord at E15.5 and E18.5. cor, cortex; di, diencephalon; drg, dorsal root ganglion; enc, epithelial lining of nasal cavity; in, interneurons; lf, primary lens fibers; lge, lateral ganglionic eminence; m, mesenchyme; nr, neural retina; rp, Rathke’s pouch. Scale bars: (A) 400 lm, (B–H) 200 lm.
E15.5, Pcdh19 expression could already be detected in the mesenchymal component of the hair follicle primordium (Fig. 4G). In more mature follicles, Pcdh19 transcription
was present both in the dermal papilla and matrix (Fig. 4H). A similar expression profile was observed in the differentiating whiskers between E12.5 and E15.5 (data
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Fig. 3. Expression of Pcdh19 in the renal system. (A) At E9.5, specific expression is detected in the nephric duct and nephric cord of the mesonephros. (B) At E10.5, Pcdh19 expression (blue) coincides with Pax2 (red-brown) in the nephric duct and mesonephric tubules. (C) Counterstaining with Pax2 (redbrown) reveals the presence of Pcdh19 (blue) in the ureter tip, collecting duct and renal vesicles of the early metanephros but absence of expression in the condensing mesenchyme at E12.5. Low levels were observed in the mesenchyme surrounding the kidney. (D) At E18.5, Pcdh19 is maintained in the ureter tip and renal vesicle and is now detectable in the comma-shaped bodies and at low levels in distal convoluted tubules. Glomeruli are negative for Pcdh19 expression. cd, collecting duct; cm, condensing mesenchyme; cb, comma-shaped body; cd, collecting duct; cm, condensing mesenchyme; dct, distal convoluted tubule; g, glomerulus; mst, mesonephric tubule; nc, nephric cord; nd, nephric duct; rv, renal vesicle; sb, s-shaped body; sm, surrounding mesenchyme; ut, ureter tip. Scale bars, 50 lm.
not shown). These analyses further revealed a diffuse expression of Pcdh19 on the mesenchymal layers beneath the skin (Fig. 4G). Altogether, these data clearly demonstrate the fine regulation of Pcdh19 in specific cell types of several tissues undergoing active morphogenesis.
et al., 2000) and the Xenopus Pcdh1, found in the brain, kidney, somite and otic vesicle (Kuroda et al., 2002). However, the expression profile described here is unique among the protocadherins documented to date. Hence, these data constitute a strong basis for elucidating the function of Pcdh19 in the developing embryo.
1.6. Conclusions In a search for potential developmental regulators of kidney development, we have identified Pcdh19, a poorly characterized member of the protocadherin family. Using in situ hybridization, we showed that Pcdh19 is expressed in a tissue-specific manner during embryogenesis. Pcdh19 was found in the epithelial compartments of the kidney, olfactory system, eye, brain and late stomach and in the mesenchymal compartment of the early stomach, dermomyotome, mesenteries, dermal papilla and presomitic mesoderm. Interestingly, in most of these tissues Pcdh19 expression is first detected at the early stages of organ patterning. The tissue distribution during embryogenesis shows some overlap with other members of the d-protocadherin family, notably with mouse and zebrafish Pcdh8 and Pcdh10, which are also expressed in the brain, olfactory epithelium and paraxial mesoderm (Hirano et al., 1999; Makarenkova et al., 2005; Murakami et al., 2005; Yamamoto
2. Experimental procedures 2.1. Mice Pax2GFP BAC transgene (#30) used to sort mesonephros-specific cells was described previously (Grote et al., 2006; Pfeffer et al., 2002). Embryos for gene expression analysis were generated by timed matings of wild-type CD-1 mice. Noon of the day of plug detection was considered as E0.5.
2.2. Microarray analysis FACS sorting of Pax2(+) mesonephric cells, linear amplification and DNA microarray analysis were described previously (Bouchard et al., 2005; Grote et al., 2006).
2.3. In situ hybridization and immunohistochemistry Dissected embryos were fixed at 4 °C for 4 h to overnight in 4% paraformaldehyde and dehydrated in methanol series for whole mount in situ hybridization or equilibrated in 30% sucrose and mounted in OCT (Tissue-Tek) for cryosectioning. In situ hybridizations on whole
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Fig. 4. Expression of Pcdh19 in other tissues. In situ hybridization for Pcdh19 on transversal sections at E12.5 (A–C), E15.5 (G) and E18.5 (D–F and H). (A) Transcription signal is detected in the dermomyotome and spinal cord. (B) Urogenital and dorsal mesenteries show diffuse mRNA signal at the level of the mesonephros. (C) Pcdh19 is detected specifically in the mesenchyme surrounding the forestomach endoderm. (D) At E18.5, staining is observed in the glandular stomach. (E) At higher magnification, the expression can be found in the glandular epithelium and mesenchyme while the luminal epithelium is negative for Pcdh19. (F) Expression in the exocrine pancreas and oesophagus. (G) Onset of Pcdh19 expression in the hair follicle primordium. (H) Strong signal is observed in the dermal papilla and matrix of the mature hair follicle. dp, dermal papilla; dm, dermomyotome; dmes, dorsal mesentery; fsm, forestomach mesenchyme; ge, glandular epithelium; gm, glandular mesenchyme; gst, glandular stomach; hfp, hair follicle primordium; hsm, hindstomach mesenchyme; le, luminal epithelium; m, mesenchyme; ma, matrix; mn, mesonephros; oes, oesophagus; pa, pancreas; sc, spinal cord; umes, urogenital mesentery. Scale bars: (A and F) 200 lm, (B–D) 100 lm, (E, G and H) 50 lm. mount and sections were performed as described (Henrique et al., 1995; Hynes et al., 2000). Pcdh19 cRNA probe was generated by RT-PCR from E17.5 kidney cDNA using the following primers: 5 0 -CACCAAGCAGAA GATTGACCGAG-3 0 and 5 0 -GCCTCCCATCCACAAGAATAGTG-3 0 .
The 986 bp PCR product, corresponding to positions 1963–2949 of the reference sequence XM_205287 (exon 1), was cloned in pGEM-T-easy and used for digoxigenin-dUTP cRNA probe synthesis, following linearization. For double stainings, in situ hybridization was performed first,
Y. Gaitan, M. Bouchard / Gene Expression Patterns 6 (2006) 893–899 followed by immunohistochemistry as described previously (Bouchard et al., 2000). Immunodetection of the rabbit anti-Pax2 antibody (1:100 dilution, Covance) was revealed with the ABC Vectastain kits (Vector labs).
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