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Distinct and common developmental expression patterns of the murine Pkd2 and Pkd1 genes
Mechanisms of Development 93 (2000) 179±183
Gene expression pattern
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Distinct and common developmental expression patterns of the murine Pkd2 and Pkd1 genes Richard Guillaume, Marie Trudel* Institut de Recherches Cliniques de Montreal, Molecular Genetics and Development, Faculte de Medecine de L'Universite de Montreal, 110 ouest avenue des Pins, Montreal, Quebec, Canada H2W1R7 Received 19 July 1999; received in revised form 12 January 2000; accepted 13 January 2000
Abstract Autosomal dominant polycystic kidney disease (ADPKD) is one of the most commonly inherited renal diseases. At least two genes, PKD2 and PKD1 are implicated in the development of this disease. Our pathogenetic studies showed that the human and murine polycystic kidney disease (PKD) involves failure to switch out of a renal developmental program. We have thus undertaken a detailed comparative expression analysis of Pkd2 and Pkd1 from the morula stage to adulthood. Pkd2 expression was detected as early as the morula and blastocyst stages as observed for Pkd1. Strong Pkd2 expression, similar to Pkd1, was displayed in all mesenchymal and cartilaginous tissues during mouse development. However major differences in Pkd2 expression in comparison to Pkd1 were identi®ed. First, in contrast to Pkd1, the neural crest cell-derived tissues displayed a low to undetectable Pkd2 expression at all ages. Second, no increase in Pkd2 expression was detected during mesenchymal condensation. Third, high Pkd2 expression in the kidneys was localized mainly to the tubular epithelium of the cortical region from murine development to adulthood. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pkd2; Pkd1; Metanephric blastema; Kidney; Mesenchyme; Cartilage
Autosomal dominant polycystic kidney disease (ADPKD) is characterized by bilateral renal cysts that initiate in utero and progressively leads to renal insuf®ciency in adulthood. In addition, extrarenal manifestations such as cardiac valvular abnormalities and cerebral aneurysms are common in ADPKD. This human disease is multigenic and involves at least three different loci: PKD1 which accounts for ~85% of all cases, PKD2 for ~15% and a third locus that remains to be identi®ed (Consortium, 1994; 1995; Burn et al., 1995; Daoust et al., 1995; Mochizuki et al., 1996). More recently, the murine Pkd1 and Pkd2 loci have been cloned (LoÈhning et al., 1997; Wu et al., 1997; Guillaume et al., 1999), both have been proposed to encode a transmembrane protein based on sequence motifs and have been called polycystin-1 and polycystin-2, respectively. Mice with the Pkd1 del 34 allele or null for the Pkd2 gene die during embryonic development but at different gestational ages (Lu et al., 1997; Wu et al., 1998). Both the polycystin-2 and polycystin-1 cytoplasmic carboxy terminal regions contain a coilcoiled domain which has been shown to interact together in vitro (Tsiokas et al., 1997; Qian et al., 1997). Toward the * Corresponding author. Tel.: 11-514-987-5712; fax: 11-514-9875585. E-mail address: [email protected] (M. Trudel)
characterization of the polycystin-2 and polycystin-1 roles and of their potential interactions, we have undertaken a detailed comparison of their expression pattern during normal mouse development.
1. Results and discussion We have initiated our expression studies at the murine morula stage. By RT-PCR, Pkd2 transcript was detectable at low levels in the morula stage (8±16 cells) and this expression appeared higher in murine ES-129 undifferentiated cells as previously observed for Pkd1 (Fig. 1A). Consistent with this ®nding, Pkd2 and Pkd1 expression was detected in isolated blastocysts by in situ hybridization (Fig. 1B±D). Expression of Pkd2 and Pkd1 was maintained after blastocyst implantation through the egg cylinder stage (e6.5) (Fig. 2). The signals were identi®ed over all the embryonic primordial layers and particularly strong signals were detected over the decidual region. Subsequently, at e9.5 most of the embryo showed pleiotropic Pkd2 and Pkd1 expression (Fig. 2). However, Pkd2 and Pkd1 expression was barely detectable in the myocardium of the embryo and weakly expressed in the myometrium of the maternal uterine muscular wall.
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R. Guillaume, M. Trudel / Mechanisms of Development 93 (2000) 179±183
Fig. 1. Pkd2 and Pkd1 expression in preimplantation embryos. (A) Pkd2 expression was demonstrated using RT-PCR analysis, in morula (Mo) and ES cells (ES) with a 216 bp fragment (*), the expression of S16 ribosomal RNA was monitored as internal control with a 103 bp fragment (arrow); C represents a negative control and M indicates a molecular weight marker of 100 bp ladder. (B) Blastocyst displayed a Pkd1 signal with the antisense Pkd1 probe. (C) Blastocyst displayed a strong signal with the antisense Pkd2 probe. (D) Same blastocyst as in (C) from adjacent section with the sense Pkd2 probe showed a background signal. Scale bar: 10 mM.
At e11.5 strong expression of Pkd1 and Pkd2 was observed in mesenchymal tissues and sclerotomes. By contrast, a difference between Pkd1 and Pkd2 expression pattern becomes apparent at this stage, where a strong signal is observed over the hindbrain, midbrain and forebrain for Pkd1 and a weaker to undetectable signal for Pkd2. At e12.5, expression of Pkd1 and Pkd2 in mesenchymal tissues is maintained in the face and limbs, and displayed in lung, intestinal/midgut loops and umbilical cord (Fig. 2). The metanephric blastema displayed a moderate and diffuse Pkd1 and Pkd2 expression signal at this stage. Highly pronounced Pkd1 and Pkd2 expression was observed in nasal and vertebral cartilaginous tissues. Although Pkd1 and Pkd2 expression is still barely detectable in the myocardium, a strong Pkd1 and Pkd2 expression signal was observed over the cardiac atrio-ventricular cushion, aortic arch and thoracic aorta (Fig. 2). At e12.5, the diaphragm was the ®rst muscle to show strong expression of both Pkd2 and Pkd1 (Fig. 2). The difference in hybridization signal revealed at e11.5 for Pkd1 and Pkd2 was sustained in the central and peripheral nervous system, namely brain, spinal cord and dorsal root ganglia. This difference was further emphasized in the posterior compartment of the central nervous system, as Pkd2 expression was low to undetectable (Fig. 2). In contrast to Pkd1 that shows increased expression upon mesenchymal condensation in the kidney, lung, salivary gland, skin (Guillaume et al., 1999), the level of expression of Pkd2 in these tissues appears not to increase during mesenchymal to epithelial conversion. Similar to Pkd1, a strong Pkd2 hybridization signal was identi®ed at e13.5 in the muscularis of the gastrointestinal loops. The muscularis of the bladder in contrast, showed a
weaker Pkd1 signal than Pkd2 at e13.5 but the signal became equivalent at e16.5. The high Pkd2 expression pattern in cartilaginous tissues was maintained at e13.5 and e15.5 and was con®ned to the pre-chondrogenic region such as in the limb cartilage until e18.5. At e18.5, Pkd2 and Pkd1 were both strongly expressed in the smooth muscle lamina propria and muscularis of the gastrointestinal loops. By this age, the lungs displayed the most intense Pkd1 signal relative to all other fetal tissues. In the kidneys, Pkd2 expression became signi®cantly stronger than that of Pkd1 at e15.5 (Figs. 2 and 3A±D). Fetuses at e15.5 also revealed a diffuse Pkd2 signal in the adrenal gland whereas the Pkd1 signal was intense in the adrenal medulla and capsule, a pattern that persisted thereafter (Fig. 3A,B). By e18.5, renal Pkd2 expression was the most intense signal of all tissues observed in these fetuses (Fig. 2). Strong and equivalent expression was observed for Pkd2 and Pkd1 in the nephrogenic cortical zone and in collecting ducts (Fig. 3E,F). Pkd2 expression was highest in cortical tubules and signi®cantly weaker in the glomeruli whereas Pkd1 was expressed at similar intensity over glomeruli and tubules. This distinct Pkd2 and Pkd1 expression pattern for glomeruli and tubules during renal development was similar to that reported by immunostaining (Grif®n et al., 1997; Markowitz et al., 1999). In adult kidneys, strongest Pkd2 expression was identi®ed over the tubular epithelium of the cortex and a signi®cantly weaker signal was detected over the glomeruli (Fig. 3H,J). This adult renal pattern of expression differed from the Pkd1 expression that showed a similar weak signal intensity over both tubules and glomeruli (Fig. 3I) and a moderate signal for collecting tubules (Fig. 3G).
Fig. 2. Expression pattern of Pkd2 and Pkd1 from e6.5 to e18.5. In situ hybridization expression analysis carried on sagittal sections at e6.5, e11.5, e12.5, e13.5, e15.5, e18.5 and frontal section at e9.5. Sections were hybridized with antisense Pkd1 probe (®rst row), with antisense Pkd2 probe (second row) and with sense Pkd2 probe (third row). The latter consists of control adjacent sections to those hybridized with antisense Pkd2 probe. e6.5: embryo (e), decidua (d), myometrium (my); e9.5: neural tube (nt), myocardium (myo); e11.5: hindbrain (hi), midbrain (mi), forebrain (fo), facial mesenchyme (fm), mandibular component of ®rst branchial arch (ma), limb mesenchyme (lm), sclerotomes (sc); e12.5: median hinge point (mhp), spinal cord (sp), vertebral cartilage primordium (vc), atrio-ventricular cushion (at), dorsal root ganglia (drg), lung (lu), neopallial cortex (nc), nasal cartilage primordium (na), diaphragm (di), metanephros (me), midgut loop (mg), cochleal cartilage (co), aortic arch and pulmonary trunk (a), thoracic aorta (ta), umbilical cord (uc); e13.5: roof of midbrain (rm), cerebellar primordium (cp), choroid plexus (ch), lateral ventricle (lv), mantle layer (ml), tongue (t), mandibular cartilage (mc), spinal ganglia (sg), intestinal mesenchyme (im), bladder (b); e15.5: cervical ganglia (cg), cricoõÈd cartilage (cr), facial cartilage (fc), limb cartilage (lc), adrenal gland (ag), kidneys (k); e18.5: follicle of vibrissae (fv), submandibular gland (su), skin (sk), perimysium (p), stomach (st), ureter (ur). Dark ®eld; scale bar: 1 mm.
R. Guillaume, M. Trudel / Mechanisms of Development 93 (2000) 179±183
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R. Guillaume, M. Trudel / Mechanisms of Development 93 (2000) 179±183
In conclusion, our studies have shown striking similarities between Pkd2 and Pkd1 expression in the early embryo and fetuses, supporting possible in vivo interactions of the two proteins toward regulation of a common developmental
pathway. Importantly, the pattern of Pkd1 and Pkd2 renal expression is consistent with a role in tubulogenic differentiation and in maintenance of mature nephron homeostasis. Our results also demonstrated spatial differences in Pkd2 and Pkd1 expression, suggesting additional independent roles for each protein. 2. Materials and methods 2.1. Morulae and ES cells RT-PCR Morulae of 8±16 cells were obtained from 2.5 days pregnant mice uteri by ¯ushing with M2 media, rinsed and immediately processed for RNA extraction and reverse transcription as previously described (Lanoix et al., 1996; Trudel et al., 1997). Undifferentiated ES 129 cells were similarly processed for RNA extraction and reverse transcription. Co-ampli®cation of the Pkd2 (forward primer 5 0 -GATTGACGCCGTGATTGTCAAG-3 0 , reverse primer and S16 5 0 -TGCTTACACCATGACCTGTTTGC-3 0 ) (forward 5 0 -AGGAGCGATTTGCTGGTGTGGA-3 0 and reverse 5 0 -GCTACCAGGCCTTTGAGATGGA-3 0 ) transcripts were carried out in the following conditions: 948C, 1 min, 948C, 10 s, 668C, 25 s, for 30 PCR cycles. Parallel control reactions were carried out with water replacing DNA. 2.2. In situ hybridization The in situ hybridization protocol was performed on mouse (C57BL/6J £ CBA/J) F2 embryos aged from blastocysts to 18.5dpc as previously described (Trudel et al., 1998; Guillaume et al., 1999). Blastocysts were obtained from 3.5±4 days pregnant mice. All tissue or embryo samples were ®xed in 4% paraformaldehyde and embedded in paraf®n. Three micron thick sections were hybridized with antisense and sense probes as a control. The murine Pkd2 probes were produced by subcloning a 933 bp EcoRVFig. 3. Renal temporal Pkd2 and Pkd1 expression. Para-sagittal e15.5 kidney sections hybridized with the antisense Pkd1 (A,C) and Pkd2 (B,D) probes, demonstrated higher Pkd2 expression than Pkd1 over cortical region (A,B) in dark ®eld or tubules (C,D) in bright ®eld. The depicted boxes in (A,B) are illustrated at higher magni®cation in (C,D) respectively, kidneys (k), adrenal capsule (ac), adrenal medulla (am), liver (li), tubules (t) mesenchyme (m). Scale bar: 100 mm. Renal sagittal e18.5 sections hybridized with antisense Pkd1 (E) and Pkd2 (F) probes showed high Pkd2 and Pkd1 expression in the nephrogenic cortical zone (cz) and collecting ducts (cd). Pkd2 was expressed highly in cortical tubules (t) and weakly in glomeruli (g) whereas a similar strong Pkd1 expression was observed in tubules and glomeruli. Scale bar: 100 mm. Adult kidney sagittal sections hybridized with antisense Pkd1 (G,I) and Pkd2 (H,J) probes, showed higher Pkd2 expression than Pkd1 in the cortical region in dark ®eld (G,H) or bright ®eld (I,J); cortex (c) and medulla (m) and illustrations (G,H) follow scale bar: 1 mm. The depicted boxes in (G,H) are illustrated at higher magni®cation in (I,J) respectively, revealed similar weak Pkd1 and Pkd2 signal over glomeruli (g) and higher Pkd2 signal over tubules (t). Scale bar: 100 mm.
R. Guillaume, M. Trudel / Mechanisms of Development 93 (2000) 179±183
PstI Pkd2 fragment (nucleotides 2006±2933) encoding the carboxy terminal PKD2 region from a Pkd2 cDNA plasmid (generous gift of Dr. S. Somlo) into pBluescript. This plasmid was linearized by EcoRV to synthesize the antisense T3 RNA probe and by SmaI for the sense T7 RNA probe. The Pkd1 antisense probe consisted of a murine 1.6 kb Pkd1 cDNA (exon 36±45) (Guillaume et al., 1999). Acknowledgements This work was supported by the MRC of Canada. References Burn, T.C., Connors, T.D., Dackowski, W.R., Petry, L.R., Van Raay, T.J., Millholland, J.M., Venet, M., Miller, G., Hakim, R.M., Landes, G.M., Klinger, K.W., Qian, F., Onuchic, L.F., Watnick, T., Germino, G.G., Doggett, N.A., 1995. Analysis of the genomic sequence for the autosomal dominant polycystic kidney disease (PKD1) gene predicts the presence of a leucine-rich repeat. Hum. Mol. Genet. 4, 575±582. Consortium, E.P.K.D., 1994. The polycystic kidney disease 1 gene encodes a 14 kb transcript and lies within a duplicated region on chromosome 16. Cell 77, 881±894. Consortium, I.P.K.D., 1995. Polycystic Kidney Disease: The complete structure of the PKD1 gene and its protein. Cell 81, 289±298. Daoust, M., Reynolds, D., Bichet, D.G., Somlo, S., 1995. Evidence for a third Genetic locus for autosomal dominant polycystic kidney disease. Genomics 25, 733±736. Grif®n, M.D., O'Sullivan, D.A., Torres, V.E., Grande, J.P., Kanwar, Y.S., Kumar, R., 1997. Expression of polycystin in mouse metanephros and extra-metanephric tissues. Kidney Int. 52, 1196±1205. Guillaume, R., D'Agati, V., Daoust, M., Trudel, M., 1999. Murine Pkd1 is a developmentally regulated gene from morula to adulthood: role in tissue condensation and patterning. Dev. Dyn. 214, 337±348.
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Lanoix, J., D'Agati, V., Szabolcs, M., Trudel, M., 1996. Dysregulation of cellular proliferation and apoptosis mediates human autosomal dominant polycystic kidney disease (ADPKD). Oncogene 13, 1153±1160. LoÈhning, C., Nowicka, U., Frischauf, A.-M., 1997. The mouse homolog of PKD1: sequence analysis and alternative splicing. Mamm. Genome 8, 307±311. Lu, W., Peissel, B., Babkhanlou, H., Pavlova, A., Geng, L., Fan, X., Larson, C., Brent, G., Zhou, J., 1997. Perinatal lethality with kidney and pancreas defects in mice with a targeted PKD1 mutation. Nat. Genet. 17, 179±181. Markowitz, G.S., Cai, Y., Li, L., Wu, G., Ward, L., Somlo, S., D'Agati, V., 1999. Polycystic-2 expression is developmentally regulated. Am. J. Physiol. (Renal Physiol. 46, F17±F25. Mochizuki, T., Wu, G., Hayashi, T., Xenophontos, S., Veldhuisen, B., Saris, J.J., Reynolds, D.M., Cai, Y., Gabow, P.A., Pierides, A., Kimberling, W.J., Breuning, M.H., Deltas, C.C., Peters, D.J.M., Somlo, S., 1996. PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein. Science 272, 1339±1342. Qian, F., Germino, F.J., Cai, Y., Zhang, X., Somlo, S., Germino, G.G., 1997. PKD1 interacts with PKD2 through a probable coiled-coil domain. Nat. Genet. 16, 179±183. Trudel, M., Lanoix, J., Barisoni, J., Blouin, M.-J., Desforges, M., L'Italien, C., 1997. D'Agati. V., C-MYC-induced Apoptosis in Polycystic Kidney Disease is Bcl-2 and p53 Independent. J. Exp. Med. 186, 1873± 1884. Trudel, M., Barisoni, L., Lanoix, J., D'Agati, V., 1998. Polycystic Kidney Disease in SBM Transgenic Mice: role of c-myc in Disease Induction and Progression. Am. J. Pathol. 152, 219±229. Tsiokas, L., Kim, E., Arnould, T., Sukhatme, V.P., Walz, G., 1997. Homoand heterodimeric interactions between the gene product of PKD1 and PKD2. Proc. Natl. Acad. Sci. USA 94, 6965±6970. Wu, G., Mochizuki, T., Le, T.C., Cai, Y., Hayashi, T., Reynolds, D.M., Somlo, S., 1997. Molecular cloning, cDNA sequence analysis, and chromosomal localization of Mouse Pkd2. Genomics 45, 220±223. Wu, Q., D'Agati, V., Cai, G., Markowitz, J.H., Park, D.M., Reynolds, Y., Maeda, T.C., Le, J.R., Hou, H., Kucherlapati, R., Edelmann, W., Somlo, S., 1998. Somatic inactivation of Pkd2 results in polycystic kidney disease. Cell 93, 177±188.
Gene expression pattern
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Distinct and common developmental expression patterns of the murine Pkd2 and Pkd1 genes Richard Guillaume, Marie Trudel* Institut de Recherches Cliniques de Montreal, Molecular Genetics and Development, Faculte de Medecine de L'Universite de Montreal, 110 ouest avenue des Pins, Montreal, Quebec, Canada H2W1R7 Received 19 July 1999; received in revised form 12 January 2000; accepted 13 January 2000
Abstract Autosomal dominant polycystic kidney disease (ADPKD) is one of the most commonly inherited renal diseases. At least two genes, PKD2 and PKD1 are implicated in the development of this disease. Our pathogenetic studies showed that the human and murine polycystic kidney disease (PKD) involves failure to switch out of a renal developmental program. We have thus undertaken a detailed comparative expression analysis of Pkd2 and Pkd1 from the morula stage to adulthood. Pkd2 expression was detected as early as the morula and blastocyst stages as observed for Pkd1. Strong Pkd2 expression, similar to Pkd1, was displayed in all mesenchymal and cartilaginous tissues during mouse development. However major differences in Pkd2 expression in comparison to Pkd1 were identi®ed. First, in contrast to Pkd1, the neural crest cell-derived tissues displayed a low to undetectable Pkd2 expression at all ages. Second, no increase in Pkd2 expression was detected during mesenchymal condensation. Third, high Pkd2 expression in the kidneys was localized mainly to the tubular epithelium of the cortical region from murine development to adulthood. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pkd2; Pkd1; Metanephric blastema; Kidney; Mesenchyme; Cartilage
Autosomal dominant polycystic kidney disease (ADPKD) is characterized by bilateral renal cysts that initiate in utero and progressively leads to renal insuf®ciency in adulthood. In addition, extrarenal manifestations such as cardiac valvular abnormalities and cerebral aneurysms are common in ADPKD. This human disease is multigenic and involves at least three different loci: PKD1 which accounts for ~85% of all cases, PKD2 for ~15% and a third locus that remains to be identi®ed (Consortium, 1994; 1995; Burn et al., 1995; Daoust et al., 1995; Mochizuki et al., 1996). More recently, the murine Pkd1 and Pkd2 loci have been cloned (LoÈhning et al., 1997; Wu et al., 1997; Guillaume et al., 1999), both have been proposed to encode a transmembrane protein based on sequence motifs and have been called polycystin-1 and polycystin-2, respectively. Mice with the Pkd1 del 34 allele or null for the Pkd2 gene die during embryonic development but at different gestational ages (Lu et al., 1997; Wu et al., 1998). Both the polycystin-2 and polycystin-1 cytoplasmic carboxy terminal regions contain a coilcoiled domain which has been shown to interact together in vitro (Tsiokas et al., 1997; Qian et al., 1997). Toward the * Corresponding author. Tel.: 11-514-987-5712; fax: 11-514-9875585. E-mail address: [email protected] (M. Trudel)
characterization of the polycystin-2 and polycystin-1 roles and of their potential interactions, we have undertaken a detailed comparison of their expression pattern during normal mouse development.
1. Results and discussion We have initiated our expression studies at the murine morula stage. By RT-PCR, Pkd2 transcript was detectable at low levels in the morula stage (8±16 cells) and this expression appeared higher in murine ES-129 undifferentiated cells as previously observed for Pkd1 (Fig. 1A). Consistent with this ®nding, Pkd2 and Pkd1 expression was detected in isolated blastocysts by in situ hybridization (Fig. 1B±D). Expression of Pkd2 and Pkd1 was maintained after blastocyst implantation through the egg cylinder stage (e6.5) (Fig. 2). The signals were identi®ed over all the embryonic primordial layers and particularly strong signals were detected over the decidual region. Subsequently, at e9.5 most of the embryo showed pleiotropic Pkd2 and Pkd1 expression (Fig. 2). However, Pkd2 and Pkd1 expression was barely detectable in the myocardium of the embryo and weakly expressed in the myometrium of the maternal uterine muscular wall.
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00257-4
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R. Guillaume, M. Trudel / Mechanisms of Development 93 (2000) 179±183
Fig. 1. Pkd2 and Pkd1 expression in preimplantation embryos. (A) Pkd2 expression was demonstrated using RT-PCR analysis, in morula (Mo) and ES cells (ES) with a 216 bp fragment (*), the expression of S16 ribosomal RNA was monitored as internal control with a 103 bp fragment (arrow); C represents a negative control and M indicates a molecular weight marker of 100 bp ladder. (B) Blastocyst displayed a Pkd1 signal with the antisense Pkd1 probe. (C) Blastocyst displayed a strong signal with the antisense Pkd2 probe. (D) Same blastocyst as in (C) from adjacent section with the sense Pkd2 probe showed a background signal. Scale bar: 10 mM.
At e11.5 strong expression of Pkd1 and Pkd2 was observed in mesenchymal tissues and sclerotomes. By contrast, a difference between Pkd1 and Pkd2 expression pattern becomes apparent at this stage, where a strong signal is observed over the hindbrain, midbrain and forebrain for Pkd1 and a weaker to undetectable signal for Pkd2. At e12.5, expression of Pkd1 and Pkd2 in mesenchymal tissues is maintained in the face and limbs, and displayed in lung, intestinal/midgut loops and umbilical cord (Fig. 2). The metanephric blastema displayed a moderate and diffuse Pkd1 and Pkd2 expression signal at this stage. Highly pronounced Pkd1 and Pkd2 expression was observed in nasal and vertebral cartilaginous tissues. Although Pkd1 and Pkd2 expression is still barely detectable in the myocardium, a strong Pkd1 and Pkd2 expression signal was observed over the cardiac atrio-ventricular cushion, aortic arch and thoracic aorta (Fig. 2). At e12.5, the diaphragm was the ®rst muscle to show strong expression of both Pkd2 and Pkd1 (Fig. 2). The difference in hybridization signal revealed at e11.5 for Pkd1 and Pkd2 was sustained in the central and peripheral nervous system, namely brain, spinal cord and dorsal root ganglia. This difference was further emphasized in the posterior compartment of the central nervous system, as Pkd2 expression was low to undetectable (Fig. 2). In contrast to Pkd1 that shows increased expression upon mesenchymal condensation in the kidney, lung, salivary gland, skin (Guillaume et al., 1999), the level of expression of Pkd2 in these tissues appears not to increase during mesenchymal to epithelial conversion. Similar to Pkd1, a strong Pkd2 hybridization signal was identi®ed at e13.5 in the muscularis of the gastrointestinal loops. The muscularis of the bladder in contrast, showed a
weaker Pkd1 signal than Pkd2 at e13.5 but the signal became equivalent at e16.5. The high Pkd2 expression pattern in cartilaginous tissues was maintained at e13.5 and e15.5 and was con®ned to the pre-chondrogenic region such as in the limb cartilage until e18.5. At e18.5, Pkd2 and Pkd1 were both strongly expressed in the smooth muscle lamina propria and muscularis of the gastrointestinal loops. By this age, the lungs displayed the most intense Pkd1 signal relative to all other fetal tissues. In the kidneys, Pkd2 expression became signi®cantly stronger than that of Pkd1 at e15.5 (Figs. 2 and 3A±D). Fetuses at e15.5 also revealed a diffuse Pkd2 signal in the adrenal gland whereas the Pkd1 signal was intense in the adrenal medulla and capsule, a pattern that persisted thereafter (Fig. 3A,B). By e18.5, renal Pkd2 expression was the most intense signal of all tissues observed in these fetuses (Fig. 2). Strong and equivalent expression was observed for Pkd2 and Pkd1 in the nephrogenic cortical zone and in collecting ducts (Fig. 3E,F). Pkd2 expression was highest in cortical tubules and signi®cantly weaker in the glomeruli whereas Pkd1 was expressed at similar intensity over glomeruli and tubules. This distinct Pkd2 and Pkd1 expression pattern for glomeruli and tubules during renal development was similar to that reported by immunostaining (Grif®n et al., 1997; Markowitz et al., 1999). In adult kidneys, strongest Pkd2 expression was identi®ed over the tubular epithelium of the cortex and a signi®cantly weaker signal was detected over the glomeruli (Fig. 3H,J). This adult renal pattern of expression differed from the Pkd1 expression that showed a similar weak signal intensity over both tubules and glomeruli (Fig. 3I) and a moderate signal for collecting tubules (Fig. 3G).
Fig. 2. Expression pattern of Pkd2 and Pkd1 from e6.5 to e18.5. In situ hybridization expression analysis carried on sagittal sections at e6.5, e11.5, e12.5, e13.5, e15.5, e18.5 and frontal section at e9.5. Sections were hybridized with antisense Pkd1 probe (®rst row), with antisense Pkd2 probe (second row) and with sense Pkd2 probe (third row). The latter consists of control adjacent sections to those hybridized with antisense Pkd2 probe. e6.5: embryo (e), decidua (d), myometrium (my); e9.5: neural tube (nt), myocardium (myo); e11.5: hindbrain (hi), midbrain (mi), forebrain (fo), facial mesenchyme (fm), mandibular component of ®rst branchial arch (ma), limb mesenchyme (lm), sclerotomes (sc); e12.5: median hinge point (mhp), spinal cord (sp), vertebral cartilage primordium (vc), atrio-ventricular cushion (at), dorsal root ganglia (drg), lung (lu), neopallial cortex (nc), nasal cartilage primordium (na), diaphragm (di), metanephros (me), midgut loop (mg), cochleal cartilage (co), aortic arch and pulmonary trunk (a), thoracic aorta (ta), umbilical cord (uc); e13.5: roof of midbrain (rm), cerebellar primordium (cp), choroid plexus (ch), lateral ventricle (lv), mantle layer (ml), tongue (t), mandibular cartilage (mc), spinal ganglia (sg), intestinal mesenchyme (im), bladder (b); e15.5: cervical ganglia (cg), cricoõÈd cartilage (cr), facial cartilage (fc), limb cartilage (lc), adrenal gland (ag), kidneys (k); e18.5: follicle of vibrissae (fv), submandibular gland (su), skin (sk), perimysium (p), stomach (st), ureter (ur). Dark ®eld; scale bar: 1 mm.
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R. Guillaume, M. Trudel / Mechanisms of Development 93 (2000) 179±183
In conclusion, our studies have shown striking similarities between Pkd2 and Pkd1 expression in the early embryo and fetuses, supporting possible in vivo interactions of the two proteins toward regulation of a common developmental
pathway. Importantly, the pattern of Pkd1 and Pkd2 renal expression is consistent with a role in tubulogenic differentiation and in maintenance of mature nephron homeostasis. Our results also demonstrated spatial differences in Pkd2 and Pkd1 expression, suggesting additional independent roles for each protein. 2. Materials and methods 2.1. Morulae and ES cells RT-PCR Morulae of 8±16 cells were obtained from 2.5 days pregnant mice uteri by ¯ushing with M2 media, rinsed and immediately processed for RNA extraction and reverse transcription as previously described (Lanoix et al., 1996; Trudel et al., 1997). Undifferentiated ES 129 cells were similarly processed for RNA extraction and reverse transcription. Co-ampli®cation of the Pkd2 (forward primer 5 0 -GATTGACGCCGTGATTGTCAAG-3 0 , reverse primer and S16 5 0 -TGCTTACACCATGACCTGTTTGC-3 0 ) (forward 5 0 -AGGAGCGATTTGCTGGTGTGGA-3 0 and reverse 5 0 -GCTACCAGGCCTTTGAGATGGA-3 0 ) transcripts were carried out in the following conditions: 948C, 1 min, 948C, 10 s, 668C, 25 s, for 30 PCR cycles. Parallel control reactions were carried out with water replacing DNA. 2.2. In situ hybridization The in situ hybridization protocol was performed on mouse (C57BL/6J £ CBA/J) F2 embryos aged from blastocysts to 18.5dpc as previously described (Trudel et al., 1998; Guillaume et al., 1999). Blastocysts were obtained from 3.5±4 days pregnant mice. All tissue or embryo samples were ®xed in 4% paraformaldehyde and embedded in paraf®n. Three micron thick sections were hybridized with antisense and sense probes as a control. The murine Pkd2 probes were produced by subcloning a 933 bp EcoRVFig. 3. Renal temporal Pkd2 and Pkd1 expression. Para-sagittal e15.5 kidney sections hybridized with the antisense Pkd1 (A,C) and Pkd2 (B,D) probes, demonstrated higher Pkd2 expression than Pkd1 over cortical region (A,B) in dark ®eld or tubules (C,D) in bright ®eld. The depicted boxes in (A,B) are illustrated at higher magni®cation in (C,D) respectively, kidneys (k), adrenal capsule (ac), adrenal medulla (am), liver (li), tubules (t) mesenchyme (m). Scale bar: 100 mm. Renal sagittal e18.5 sections hybridized with antisense Pkd1 (E) and Pkd2 (F) probes showed high Pkd2 and Pkd1 expression in the nephrogenic cortical zone (cz) and collecting ducts (cd). Pkd2 was expressed highly in cortical tubules (t) and weakly in glomeruli (g) whereas a similar strong Pkd1 expression was observed in tubules and glomeruli. Scale bar: 100 mm. Adult kidney sagittal sections hybridized with antisense Pkd1 (G,I) and Pkd2 (H,J) probes, showed higher Pkd2 expression than Pkd1 in the cortical region in dark ®eld (G,H) or bright ®eld (I,J); cortex (c) and medulla (m) and illustrations (G,H) follow scale bar: 1 mm. The depicted boxes in (G,H) are illustrated at higher magni®cation in (I,J) respectively, revealed similar weak Pkd1 and Pkd2 signal over glomeruli (g) and higher Pkd2 signal over tubules (t). Scale bar: 100 mm.
R. Guillaume, M. Trudel / Mechanisms of Development 93 (2000) 179±183
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