Abstract
In order to investigate developmental processes, several methods have been established that allow the visualization of local proliferation zones and to follow their dynamics during morphogenesis. In this study we present a detailed description of transitory and continuous proliferation zones in the developing chick embryo. By tracing the S-phase marker proliferating cell nuclear antigen (PCNA) at the mRNA level we were able to identify the initiation and termination of proliferation programs. This approach provides additional information in comparison to the well-known BrdU incorporation or the PCNA immunostaining, which exclusively labels cells that contain PCNA protein. By means of PCNA in situ hybridization we analyzed the normal expression pattern in the 2- to 5-day-old chick embryo. We furthermore monitored the effects on PCNA expression after various manipulations such as removal of the apical ectodermal ridge (AER), the zone of polarizing activity (ZPA), and the surface ectoderm. In addition, we applied morphogens, such as fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), and retinoic acid (RA), and subsequently analyzed changes in the pattern of PCNA expression. While ablation of ZPA, AER, or ectoderm are known to reduce cell proliferation and were paralleled by loss of PCNA expression, neither BMP-2 nor BMP-4 affected PCNA expression. Upregulation of PCNA expression could be achieved by application of RA or FGFs, factors known to induce cell proliferation during limb bud outgrowth. The PCNA in situ hybridization data presented here clearly show that this method offers a novel, very sensitive tool for tracing cell proliferation and for visualizing the dynamic patterns arising due to the initiation and termination of the proliferation program.
Similar content being viewed by others
References
Amthor H, Christ B, Weil M, Patel K (1998) The importance of timing differentiation during limb muscle development. Curr Biol 8:642–652
Aulthouse AL, Solursh M (1987) The detection of a precartilage, blastema-specific marker. Dev Biol 120:377–384
Bellacosa A (2001) Functional interactions and signaling properties of mammalian DNA mismatch repair proteins. Cell Death Differ 8:1076–1092
Bosch FX, Udvarhelyi N, Venter E (1993) Expression of the histone H3 gene in benign semi-malignant lesions of the head and neck: a reliable proliferation marker. Eur J Cancer 29A:1454–1461
Bruck I, O’Donnel M (2001) The ring-type polymerase sliding clamp family. Genome Biol 2:3001.1–3001.3
Capdevila J, Izpisua-Belmonte JC (2001) Patterning mechanisms controlling vertebrate limb development. Annu Rev Cell Dev Biol 17:87–132
Chang C-D, Ottavio L, Travali S, Lipson KE, Baserga R (1990) Transcriptional and posttranscriptional regulation of the proliferating cell nuclear antigen gene. Mol Cell Biol 10:3289–3296
Chou MY, Chang ALC, McBride J, Donoff B, Gallagher GT, Wong DTW (1990) A rapid method to determine proliferation patterns of normal and malignant tissues by H3 mRNA in situ hybridization. Am J Pathol 136:729–733
Cossmann PH, Eggli PS, Kurz H (2000) Three-dimensional analysis of DNA replication foci: a comparative study on species and cell type in situ. Histochem Cell Biol 113:195–205
Delfini MC, Hirsinger E, Pourquie O, Duprez D (2000) Delta 1-activated Notch inhibits muscle differentiation without affecting Myf5 and Pax3 expression in chick limb myogenesis. Development 127:5213–5224
Dessau W, von der Mark H, von der Mark K, Fischer S (1980) Changes in the patterns of collagens and fibronectin during limb-bud chondrogenesis. J Embryol Exp Morphol 57:51–60
Francis PH, Richardson MK, Brickell PM, Tickle C (1994) Bone morphogenetic proteins and a signaling pathways that controls patterning in the developing chick limb. Development 120:209–218
Francis-West PH, Robertson K, Ede DA, Rodriguez C, Izpisua-Belmonte JC, Houston B, Burt DW, Gribbin C, Brickell PM, Tickle C (1995) Expression of genes encoding bone morphogenetic proteins and sonic hedgehog in talpid (ta3) limb buds: their relationships in the signaling cascade involved in limb patterning. Dev Dyn 203:187–197
Geduspan JS, MacCabe JA (1989) Transfer of dorsoventral information from mesoderm to ectoderm at the onset of limb development. Anat Rec 224:79–87
Goz B, Prusoff WH (1974/1975) Halogenated pyrimidine deoxyribonuclosides. In: Sartorelli AC, Johns DG (ed) Antineoplastic and immunosuppressive agents. Springer, New York, pp 273–347
Hall PA, Woods AL (1990) Immunohistochemical markers of cellular proliferation: achievements, problems and prospects. Cell Tissue Kinet 23:505–522
Hamburger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo. J Morphol 88:49–92
Kulyk WM, Upholt WB, Kosher RA (1989) Fibronectin gene expression during limb cartilage differentiation. Development 106:449–455
Kurki P, Ogata K, Tan EM (1988) Monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin as probes for proliferating cells by immunofluorescence microscopy and flow cytometry. J Immunol Methods 109:49–59
Laufer E, Nelson CE, Johnson RL, Morgan BA, Tabin C (1994) Sonic hedgehog and Fgf-4 act through a signaling cascade and feedback loop to integrate growth and patterning of the developing limb bud. Cell 79:993–1003
Lin MI, Das I, Schwart GM, Tsoulfas P, Mikawa T, Hempstead BL (2000) Trk C receptor signaling regulates cardiac myocyte proliferation during early heart development in vivo. Dev Biol 226:180–191
Loor G, Zhang SJ, Zhang P, Toomey NL, Lee M (1997) Identification of DNA replication and cell cycle proteins that interact with PCNA. Nucleic Acids Res 25:5041–5046
Mahmood R, Bresnick J, Hornbruch A, Mahony C, Morton N, Colquhoun K, Martin P, Lumsden A, Dickson C, Mason I (1995) A role for FGF-8 in the initiation and maintenance of vertebrate limb bud outgrowth. Curr Biol 5:797–806
Mariani FV, Martin GR (2003) Deciphering skeletal patterning: clues from the limb. Nature 423:319–325
Mercador N, Leonardo E, Piedra ME, Martinez AC, Ros MA, Torres M (2000) Opposing RA and FGF signals control proximodistal vertebrate limb development through regulation of Meis genes. Development 127:3961–3970
Mohammadi M, McMahon G, Sun L, Tang C, Hirth P, Yeh BK, Hubbard SR, Schlessinger J (1997) Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors. Science 276:955–960
Motobu M, El-Abasy M, Na K, Hirota Y (2002) Detection of mitogen-induced lymphocyte proliferation by bromodeoxyuridine (BrdU) incorporation in the chicken. J Vet Med Sci 64:377–379
Muskhelishvili L, Latendresse JR, Kodell RL, Henderson EB (2003) Evaluation of cell proliferation in rat tissues with BrdU, PCNA, Ki-67(MIB-5) immunohistochemistry and in situ hybridization for histone mRNA. J Histochem Cytochem 51:1681–1688
Nieto MA, Patel K, Wilkinson DG (1996) In situ hybridization analysis of chick embryos in whole mount and tissue sections. Methods Cell Biol 51:219–235
Niswander L (2003) Pattern formation: old models out on a limb. Nat Rev Genet 4:133–143
Niswander L, Tickle C, Vogel A, Booth I, Martin GR (1993) FGF-4 replaces the apical ectodermal ridge and directs outgrowth and patterning of the limb. Cell 75:579–587
Niswander L, Tickle C, Vogel A, Martin G (1994a) Function of FGF-4 in limb development. Mol Reprod Dev 39:83–89
Niswander L, Jeffrey S, Martin GR, Tickle C (1994b) A positive feedback loop coordinates growth and patterning in the vertebrate limb. Nature 371:609–612
Olson EN (1992) Interplay between proliferation and differentiation within the myogenic lineage. Dev Biol 154:261–272
Ordahl CP, Berdougo E, Venters SJ, Denetclaw WF (2001) The dermomyotome dorsomedial lip drives growth and morphogenesis of both the primary myotome and dermomyotome epithelium. Development 128:1731–1744
Paunesku T, Mittal S, Protic M, Oryhon J, Korolev SV, Joachimiak A, Woloschak GE (2001) Proliferating cell nuclear antigen (PCNA): ringmaster of the genome. Int J Radiat Biol 77:1007–1021
Qin LX, Tang ZY (2002) The prognostic molecular markers in hepatocellular carcinoma. World J Gastroenterol 8:385–392
Riddle RD, Johnson RL, Laufer E, Tabin C (1993) Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75:1401–1416
Sanders EJ, Varedi M, French AS (1993) Cell proliferation in the gastrulating chick embryo: a study using BrdU incorporation and PCNA localization. Development 118:389–399
Saunders JW (1948) The proximo-distal sequence of origin of the parts of the chick wing and the role of the ectoderm. J Exp Zool 108:363–402
Savage MP, Fallon JF (1995) FGF-2 mRNA and its antisense message are expressed in a developmentally specific manner in the chick limb bud and mesonephros. Dev Dyn 202:343–353
Scaal M, Pröls F, Füchtbauer E-M, Patel K, Hornik C, Köhler T, Christ B, Brand-Saberi B (2002) BMPs induce dermal markers and ectopic feather tracts. Mech Dev 110:51–60
Shirvan A, Ziv I, Machlin T, Zilkha-Falb R, Melamed E, Barzilai A (1997) Two waves of cyclin B and proliferating cell nuclear antigen expression during dopamine-triggered neuronal apoptosis. J Neurochem 69:539–549
Slowinski J, Mazurek U, Bierzynska-Macyszyn G (2002) Histone mRNA in situ hybridization in assessing proliferative activity of normal and malignant cells. Folia Histochem Cytobiol 40:335–339
Sun X, Mariani FV, Martin GR (2002) Functions of FGF signaling from the apical ectodermal ridge in limb development. Nature 418:501–508
Tickle C, Münsterberg A (2001) Vertebrate limb development: the early stages in chick and mouse. Curr Opin Genet Dev 11:476–481
Vogel A, Tickle C (1993) FGF-4 maintains polarizing activity of posterior limb bud cells in vivo and in vitro. Development 119:199–206
Williams BA, Ordahl CP (1994) Pax-3 expression in segmental mesoderm marks early stages in myogenic cell specification. Development 120:785–796
Acknowledgements
We thank Ulrike Pein, Ellen Gimbel, and Meike Ast-Dumbach for excellent technical assistance. This work was supported by the DFG (Br 957/5-3; SFB 592/B4).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Köhler, T., Pröls, F. & Brand-Saberi, B. PCNA in situ hybridization: a novel and reliable tool for detection of dynamic changes in proliferative activity. Histochem Cell Biol 123, 315–327 (2005). https://doi.org/10.1007/s00418-004-0730-9
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00418-004-0730-9