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Expression of Pcdh15 in the inner ear, nervous system and various epithelia of the developing embryo
Mechanisms of Development 105 (2001) 163±166
www.elsevier.com/locate/modo
Gene expression pattern
Expression of Pcdh15 in the inner ear, nervous system and various epithelia of the developing embryo Crystal L. Murcia a, Richard P. Woychik b,c,* a
b
Department of Genetics, Case Western Reserve University, Cleveland, OH 44106, USA Department of Pediatrics, Rainbow Babies and Children's Hospital, Case Western Reserve University, Cleveland, OH 44106, USA c Lynx Therapeutics, Hayward, CA 94545, USA Received 18 December 2000; received in revised form 23 March 2001; accepted 20 April 2001
Abstract We previously determined that Protocadherin 15 (Pcdh15) is associated with the Ames waltzer mutation in the mouse. Here we describe where the Pcdh15 gene is expressed at speci®c times during mouse development using RNA in situ hybridization. The expression of Pcdh15 is found in the sensory epithelium in the developing inner ear, in Rathke's pouch, and broadly throughout the brain with the highest level of expression being detected at embryonic day 16 (E16). Pcdh15 transcripts are also found in the developing eye, dorsal root ganglion, and the dorsal aspect of the neural tube, ¯oor plate and ependymal cells adjacent to the neural canal. Additionally, expression is also detected in the developing glomeruli of the kidney, surface of the tongue, vibrissae, bronchi of the lung, and in the epithelium of the olfactory apparatus, gut and lung. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ames waltzer; Deafness; Cadherin; Protocadherin; Cell adhesion molecule; Pcdh15
Pcdh15 is a member of the cadherin superfamily of calcium-dependant cell adhesion molecules. Four different alleles of Ames Waltzer (av) have mutations in Pcdh15 that cause deafness and vestibular defects in mice (Alagramam et al., 2001). The stereocilia of the sensory hair cells appear disorganized at 10 days after birth in av homozygotes, suggesting that establishment or maintenance of planar polarity may be abnormal in these animals (Alagramam et al., 2000; Eaton, 1997). We previously described expression of Pcdh15 in the adult inner ear, brain, spleen, kidney, female reproductive tract and heart using the northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) procedures (Alagramam et al., 2001). Many of the protocadherins that have been characterized in vertebrates are predominantly expressed in the central nervous system in adults with low or no expression in other tissues (Sano et al., 1993; Strehl et al., 1998; Hirano et al., 1999). Here we report RNA in situ analysis of Pcdh15 expression during embryogenesis in an attempt to better understand the role of this gene during development.
* Corresponding author. Tel.: 11-510-670-9488; fax: 11-510-670-9458. E-mail address: [email protected] (R.P. Woychik).
1. Results We examined expression of Pcdh15 in a variety of tissues beginning at E12. We have not yet assessed whether Pcdh15 is expressed earlier in development. In the inner ear, expression was detected in the epithelium of the cochlear duct at E12, E14 (data not shown) and E16 (Fig. 1A). Additionally, beginning at E14 (Fig. 1B) and continuing through E16 (data not shown), the semi-circular canals are positive for Pcdh15 mRNA expression. Expression of Pcdh15 is also apparent in the embryonic brain. At the earliest time point studied (E12), expression is uniform throughout the brain (data not shown). By E14, expression can be detected throughout the brain with the highest level of expression in the developing cerebellum and the cell layer adjacent to the ventricles (Fig. 1C,D,F). Expression decreases and becomes more heterogeneous farther from the ventricles (Fig. 1C,D). Expression of Pcdh15 is also found in the dorsal root ganglion at E12 (data not shown) and in the dorsal aspect of the neural tube, ¯oor plate and ependymal cells adjacent to the neural canal at E14 (Fig. 1E). Additionally, Pcdh15 expression is also apparent in Rathke's pouch, which will develop into the pituitary gland (Fig. 1H). At E16, expression is high throughout the brain, with a more de®ned gradient that decreases from the cells of the ventricular zone to those of the neopallial cortex
0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(01)00388-4
164
C.L. Murcia, R.P. Woychik / Mechanisms of Development 105 (2001) 163±166
Fig. 1. Expression of Pcdh15 in sensory epithelia and CNS development. In situ hybridization reveals expression in sensory epithelia of the cochlea (A), semicircular canals (B) and eye (J, arrow denotes pigment layer of the retina). Pcdh15 is also found throughout the developing brain, along the ventricles (arrow in D,I), in the embryonic and adult cerebellum (F,G) and in Rathke's pouch (arrow in H, asterisk indicates diencephalon). The E14 neural tube is also positive for Pcdh15 expression (E, arrow indicates ¯oor plate). ce, cerebellum; cp, choroid plexus; gc, internal granule cell layer; nc, neopallial cortex; vz, ventricular zone. Magni®cation: 200£ in A,B, 25£ in C,G and 100£ in D±F,H±J.
(Fig. 1I). In the adult CNS, Pcdh15 expression is limited to only the granule cells of the cerebellum (Fig. 1G). The developing mouse eye shows continuous expression of Pcdh15 in the neural and pigment layers of the retina and lens capsule epithelium at E12 (data not shown), E14 (Fig. 1J) and E16 (data not shown). (These in situ experiments were performed in wild-type FVB animals that are albino and, therefore, lack eye pigmentation).
Finally, expression of Pcdh15 can be found during organogenesis in a number of tissues. In the kidney, the highest level of Pcdh15 is detected in metanephric vesicles and developing glomeruli at E12 (data not shown) and E14 (Fig. 2A), and a low level of expression was detected in the metanephric mesenchyme. This localized expression in the glomeruli is maintained in the adult kidney (data not shown). At E14, Pcdh15 expression can be detected
C.L. Murcia, R.P. Woychik / Mechanisms of Development 105 (2001) 163±166
165
Fig. 2. Expression of Pcdh15 in the developing kidney (A), gut (B), follicle of the vibrissae (C), bronchi of the lung (D), olfactory epithelia (E) and the tongue (F, arrow indicates dorsal surface). The arrow in E points to the developing serous gland. m, muscle; e, epithelia; oe, olfactory epithelia. All images at 100£ magni®cation.
throughout the gastrointestinal tract in both epithelia and the surrounding muscle (Fig. 2B). Pcdh15 is also found in the follicle of the vibrissae, pulmonary bronchi, olfactory epithelium and nasal cartilage as well as in the tongue (Fig 2C±F). 2. Discussion In summary, Pcdh15 is expressed in the developing sensory epithelia of the inner ear, CNS, and epithelia of the kidney, lung and gastrointestinal tract. The broad expression of Pcdh15 during embryogenesis and the fact that it continues to be expressed in adult structures outside the CNS distinguishes it from other known protocadherins. It is noteworthy that many of the cells that express Pcdh15 during embryonic development are ciliated cells that comprise many of the polarized epithelial structures in the embryo, including those of the inner ear. 3. Methods RNA in situ hybridization experiments utilizing digoxigenin-labeled riboprobes in tissue sections were performed essentially as previously described (Hendricks et al., 1999). Dig-Pcdh15 sense and antisense probes were synthesized
with digoxigenin-11-UTP (Roche, Burlington, NC) from a 1089 bp fragment of the Pcdh15 full-length cDNA (Alagramam et al., 2001). Tissues were ®xed in 4% paraformaldehyde, cryoprotected in 20% sucrose and cryostat sectioned at 12 mm. Sections were hybridized overnight at 658C with DIG-Pcdh15 probe diluted 1:400 in hybridization buffer (50% formamide, 5£ SSC, 2% BM blocking agent (Roche), 0.1% Triton X-100, 0.1% CHAPS, 1 mg/ml tRNA, 5 mM EDTA pH 8.0, 50 mg/ml heparin). Diglabeled Pcdh15 probe was detected with BM purple AP substrate solution (Roche) for up to 1 day in the dark. Sense probes reproducibly showed no signal above background. Acknowledgements We would like to thank Dr Noel Murcia for all of his assistance throughout these experiments and for critical reading of the manuscript. Additionally, we are grateful to Dr Ellis Avner, Dr Beverly Dahms and Dr Karl Herrup for helpful discussions and for their assistance in carefully evaluating the expression data. We also thank Christine Halishak for assistance with the RNA in situ hybridization procedure. This research was supported by a grant from NIDCD grant 1RO1-DC03420 to RPW and CLM was supported by NIH training grant HD07104.
166
C.L. Murcia, R.P. Woychik / Mechanisms of Development 105 (2001) 163±166
References Alagramam, K.N., Zahorsky-Reeves, J., Wright, C.G., Pawlowski, K.S., Erway, L.C., Stubbs, L., Woychik, R.P., 2000. Neuroepithelial defects of the inner ear in a new allele of the mouse mutation Ames waltzer. Hear. Res. 148, 181±191. Alagramam, K.N., Murcia, C.L., Kwon, H.Y., Pawlowski, K.S., Wright, C.G., Woychik, R.P., 2001. The mouse Ames Waltzer hearing-loss mutant is caused by mutation of Pcdh15, a new protocadherin gene. Nat. Genet. 27, 99±102. Eaton, S., 1997. Planar polarization of Drosophila and vertebrate epithelia. Curr. Opin. Cell Biol. 9, 860±866. Hendricks, T., Francis, N., Fyodorov, D., Deneris, E.S., 1999. The ETS
domain factor Pet-1 is an early and precise marker of central serotonin neurons and interacts with a conserved element in serotonergic genes. J. Neurosci. 19 (23), 10348±10356. Hirano, S., Yan, Q., Suzuki, S., 1999. Expression of a novel protocadherin, OL-protocadherin, in a subset of functional systems in the developing mouse brain. J. Neurosci. 19 (3), 995±1005. Sano, K., Tanihara, H., Heimark, R.L., Obata, S., Davidson, M., St. John, T., Taketani, S., Suzuki, S., 1993. Protocadherins: a large family of cadherin-related molecules in central nervous system. EMBO J. 12 (6), 2249±2256. Strehl, S., Glatt, K., Liu, Q.M., Glatt, H., Lalande, M., 1998. Characterization of two novel protocadherins (PCDH8 and PCDH9) localized on human chromosome 13 and mouse chromosome 14. Genomics 53, 81±89.
www.elsevier.com/locate/modo
Gene expression pattern
Expression of Pcdh15 in the inner ear, nervous system and various epithelia of the developing embryo Crystal L. Murcia a, Richard P. Woychik b,c,* a
b
Department of Genetics, Case Western Reserve University, Cleveland, OH 44106, USA Department of Pediatrics, Rainbow Babies and Children's Hospital, Case Western Reserve University, Cleveland, OH 44106, USA c Lynx Therapeutics, Hayward, CA 94545, USA Received 18 December 2000; received in revised form 23 March 2001; accepted 20 April 2001
Abstract We previously determined that Protocadherin 15 (Pcdh15) is associated with the Ames waltzer mutation in the mouse. Here we describe where the Pcdh15 gene is expressed at speci®c times during mouse development using RNA in situ hybridization. The expression of Pcdh15 is found in the sensory epithelium in the developing inner ear, in Rathke's pouch, and broadly throughout the brain with the highest level of expression being detected at embryonic day 16 (E16). Pcdh15 transcripts are also found in the developing eye, dorsal root ganglion, and the dorsal aspect of the neural tube, ¯oor plate and ependymal cells adjacent to the neural canal. Additionally, expression is also detected in the developing glomeruli of the kidney, surface of the tongue, vibrissae, bronchi of the lung, and in the epithelium of the olfactory apparatus, gut and lung. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ames waltzer; Deafness; Cadherin; Protocadherin; Cell adhesion molecule; Pcdh15
Pcdh15 is a member of the cadherin superfamily of calcium-dependant cell adhesion molecules. Four different alleles of Ames Waltzer (av) have mutations in Pcdh15 that cause deafness and vestibular defects in mice (Alagramam et al., 2001). The stereocilia of the sensory hair cells appear disorganized at 10 days after birth in av homozygotes, suggesting that establishment or maintenance of planar polarity may be abnormal in these animals (Alagramam et al., 2000; Eaton, 1997). We previously described expression of Pcdh15 in the adult inner ear, brain, spleen, kidney, female reproductive tract and heart using the northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) procedures (Alagramam et al., 2001). Many of the protocadherins that have been characterized in vertebrates are predominantly expressed in the central nervous system in adults with low or no expression in other tissues (Sano et al., 1993; Strehl et al., 1998; Hirano et al., 1999). Here we report RNA in situ analysis of Pcdh15 expression during embryogenesis in an attempt to better understand the role of this gene during development.
* Corresponding author. Tel.: 11-510-670-9488; fax: 11-510-670-9458. E-mail address: [email protected] (R.P. Woychik).
1. Results We examined expression of Pcdh15 in a variety of tissues beginning at E12. We have not yet assessed whether Pcdh15 is expressed earlier in development. In the inner ear, expression was detected in the epithelium of the cochlear duct at E12, E14 (data not shown) and E16 (Fig. 1A). Additionally, beginning at E14 (Fig. 1B) and continuing through E16 (data not shown), the semi-circular canals are positive for Pcdh15 mRNA expression. Expression of Pcdh15 is also apparent in the embryonic brain. At the earliest time point studied (E12), expression is uniform throughout the brain (data not shown). By E14, expression can be detected throughout the brain with the highest level of expression in the developing cerebellum and the cell layer adjacent to the ventricles (Fig. 1C,D,F). Expression decreases and becomes more heterogeneous farther from the ventricles (Fig. 1C,D). Expression of Pcdh15 is also found in the dorsal root ganglion at E12 (data not shown) and in the dorsal aspect of the neural tube, ¯oor plate and ependymal cells adjacent to the neural canal at E14 (Fig. 1E). Additionally, Pcdh15 expression is also apparent in Rathke's pouch, which will develop into the pituitary gland (Fig. 1H). At E16, expression is high throughout the brain, with a more de®ned gradient that decreases from the cells of the ventricular zone to those of the neopallial cortex
0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(01)00388-4
164
C.L. Murcia, R.P. Woychik / Mechanisms of Development 105 (2001) 163±166
Fig. 1. Expression of Pcdh15 in sensory epithelia and CNS development. In situ hybridization reveals expression in sensory epithelia of the cochlea (A), semicircular canals (B) and eye (J, arrow denotes pigment layer of the retina). Pcdh15 is also found throughout the developing brain, along the ventricles (arrow in D,I), in the embryonic and adult cerebellum (F,G) and in Rathke's pouch (arrow in H, asterisk indicates diencephalon). The E14 neural tube is also positive for Pcdh15 expression (E, arrow indicates ¯oor plate). ce, cerebellum; cp, choroid plexus; gc, internal granule cell layer; nc, neopallial cortex; vz, ventricular zone. Magni®cation: 200£ in A,B, 25£ in C,G and 100£ in D±F,H±J.
(Fig. 1I). In the adult CNS, Pcdh15 expression is limited to only the granule cells of the cerebellum (Fig. 1G). The developing mouse eye shows continuous expression of Pcdh15 in the neural and pigment layers of the retina and lens capsule epithelium at E12 (data not shown), E14 (Fig. 1J) and E16 (data not shown). (These in situ experiments were performed in wild-type FVB animals that are albino and, therefore, lack eye pigmentation).
Finally, expression of Pcdh15 can be found during organogenesis in a number of tissues. In the kidney, the highest level of Pcdh15 is detected in metanephric vesicles and developing glomeruli at E12 (data not shown) and E14 (Fig. 2A), and a low level of expression was detected in the metanephric mesenchyme. This localized expression in the glomeruli is maintained in the adult kidney (data not shown). At E14, Pcdh15 expression can be detected
C.L. Murcia, R.P. Woychik / Mechanisms of Development 105 (2001) 163±166
165
Fig. 2. Expression of Pcdh15 in the developing kidney (A), gut (B), follicle of the vibrissae (C), bronchi of the lung (D), olfactory epithelia (E) and the tongue (F, arrow indicates dorsal surface). The arrow in E points to the developing serous gland. m, muscle; e, epithelia; oe, olfactory epithelia. All images at 100£ magni®cation.
throughout the gastrointestinal tract in both epithelia and the surrounding muscle (Fig. 2B). Pcdh15 is also found in the follicle of the vibrissae, pulmonary bronchi, olfactory epithelium and nasal cartilage as well as in the tongue (Fig 2C±F). 2. Discussion In summary, Pcdh15 is expressed in the developing sensory epithelia of the inner ear, CNS, and epithelia of the kidney, lung and gastrointestinal tract. The broad expression of Pcdh15 during embryogenesis and the fact that it continues to be expressed in adult structures outside the CNS distinguishes it from other known protocadherins. It is noteworthy that many of the cells that express Pcdh15 during embryonic development are ciliated cells that comprise many of the polarized epithelial structures in the embryo, including those of the inner ear. 3. Methods RNA in situ hybridization experiments utilizing digoxigenin-labeled riboprobes in tissue sections were performed essentially as previously described (Hendricks et al., 1999). Dig-Pcdh15 sense and antisense probes were synthesized
with digoxigenin-11-UTP (Roche, Burlington, NC) from a 1089 bp fragment of the Pcdh15 full-length cDNA (Alagramam et al., 2001). Tissues were ®xed in 4% paraformaldehyde, cryoprotected in 20% sucrose and cryostat sectioned at 12 mm. Sections were hybridized overnight at 658C with DIG-Pcdh15 probe diluted 1:400 in hybridization buffer (50% formamide, 5£ SSC, 2% BM blocking agent (Roche), 0.1% Triton X-100, 0.1% CHAPS, 1 mg/ml tRNA, 5 mM EDTA pH 8.0, 50 mg/ml heparin). Diglabeled Pcdh15 probe was detected with BM purple AP substrate solution (Roche) for up to 1 day in the dark. Sense probes reproducibly showed no signal above background. Acknowledgements We would like to thank Dr Noel Murcia for all of his assistance throughout these experiments and for critical reading of the manuscript. Additionally, we are grateful to Dr Ellis Avner, Dr Beverly Dahms and Dr Karl Herrup for helpful discussions and for their assistance in carefully evaluating the expression data. We also thank Christine Halishak for assistance with the RNA in situ hybridization procedure. This research was supported by a grant from NIDCD grant 1RO1-DC03420 to RPW and CLM was supported by NIH training grant HD07104.
166
C.L. Murcia, R.P. Woychik / Mechanisms of Development 105 (2001) 163±166
References Alagramam, K.N., Zahorsky-Reeves, J., Wright, C.G., Pawlowski, K.S., Erway, L.C., Stubbs, L., Woychik, R.P., 2000. Neuroepithelial defects of the inner ear in a new allele of the mouse mutation Ames waltzer. Hear. Res. 148, 181±191. Alagramam, K.N., Murcia, C.L., Kwon, H.Y., Pawlowski, K.S., Wright, C.G., Woychik, R.P., 2001. The mouse Ames Waltzer hearing-loss mutant is caused by mutation of Pcdh15, a new protocadherin gene. Nat. Genet. 27, 99±102. Eaton, S., 1997. Planar polarization of Drosophila and vertebrate epithelia. Curr. Opin. Cell Biol. 9, 860±866. Hendricks, T., Francis, N., Fyodorov, D., Deneris, E.S., 1999. The ETS
domain factor Pet-1 is an early and precise marker of central serotonin neurons and interacts with a conserved element in serotonergic genes. J. Neurosci. 19 (23), 10348±10356. Hirano, S., Yan, Q., Suzuki, S., 1999. Expression of a novel protocadherin, OL-protocadherin, in a subset of functional systems in the developing mouse brain. J. Neurosci. 19 (3), 995±1005. Sano, K., Tanihara, H., Heimark, R.L., Obata, S., Davidson, M., St. John, T., Taketani, S., Suzuki, S., 1993. Protocadherins: a large family of cadherin-related molecules in central nervous system. EMBO J. 12 (6), 2249±2256. Strehl, S., Glatt, K., Liu, Q.M., Glatt, H., Lalande, M., 1998. Characterization of two novel protocadherins (PCDH8 and PCDH9) localized on human chromosome 13 and mouse chromosome 14. Genomics 53, 81±89.