Skip to main content Accessibility help
  • Print publication year: 2007
  • Online publication date: August 2009

7 - In situ analysis of microRNA expression during vertebrate development

from II - MicroRNA functions and RNAi-mediated pathways
    • By Diana K. Darnell, Department of Cell Biology and Anatomy University of Arizona PO Box 245044 Life Sciences North 462 1501 N. Campbell Avenue Tucson, AZ 85724-5044 USA, Stacey Stanislaw, Department of Cell Biology and Anatomy University of Arizona PO Box 245044 Life Sciences North 462 1501 N. Campbell Avenue Tucson, AZ 85724-5044 USA, Simran Kaur, Department of Cell Biology and Anatomy University of Arizona PO Box 245044 Life Sciences North 462 1501 N. Campbell Avenue Tucson, AZ 85724-5044 USA, Tatiana A. Yatskievych, Department of Cell Biology and Anatomy University of Arizona PO Box 245044 Life Sciences North 462 1501 N. Campbell Avenue Tucson, AZ 85724-5044 USA, Sean Davey, Department of Cell Biology and Anatomy University of Arizona PO Box 245044 Life Sciences North 462 1501 N. Campbell Avenue Tucson, AZ 85724-5044 USA, Jay H. Konieczka, Department of Cell Biology and Anatomy University of Arizona PO Box 245044 Life Sciences North 462 1501 N. Campbell Avenue Tucson, AZ 85724-5044 USA, Parker B. Antin, Department of Cell Biology and Anatomy University of Arizona PO Box 245044 Life Sciences North 462 1501 N. Campbell Avenue Tucson, AZ 85724-5044 USA
  • Edited by Krishnarao Appasani
  • Foreword by Sidney Altman, Victor R. Ambros
  • Publisher: Cambridge University Press
  • DOI:
  • pp 102-114



A widespread class of non-coding, regulatory RNAs has been recently characterized. Because of their short length (21–22 nucleotides) they are called microRNA or miRNA. These miRNAs have been identified in diverse organisms (prokaryote, eukaryote, vertebrates, invertebrates, plants, fungi) and in viruses (Tuschl et al., 1999; Elbashir et al., 2001; Griffiths-Jones, 2004; Berezikov and Plasterk, 2005; Griffiths-Jones et al., 2006). Their apparently ancient function is to regulate specific protein concentration by inhibiting the first step of translation or by inducing specific mRNA degradation by 3′ UTR binding (He and Hannon, 2004; Pillai, 2005; Valencia-Sanchez et al., 2006). Both molecular and bioinformatics tools have been used to identify candidate miRNAs and their target mRNAs. Based on the numbers generated in these studies, it is estimated that vertebrate genomes may contain hundreds of miRNA genes that may regulate stability or translation of approximately one quarter of all mRNAs (Bentwich et al., 2005; Berezikov and Plasterk, 2005; Legendre et al., 2005; Xie et al., 2005).

Disruption of miRNA function often produces aberrations of important processes including organogenesis, and cell diversification, proliferation, and survival (Reinhart et al., 2000; Brennecke et al., 2003; Dostie et al., 2003; Ambros, 2004; Calin et al., 2004; Alvarez-Garcia and Miska, 2005; Giraldez et al., 2005). The miRNA function has also been implicated in regulating stem cell renewal and the onset of certain cancers (Hatfield et al., 2005, Lu et al., 2005). Therefore, miRNAs regulate important processes in animal development, physiology and disease.

Alvarez-Garcia, I. and Miska, E. A. (2005). MicroRNA functions in animal development and human disease. Development, 132, 4653–4662.
Ambros, V. (2004). The functions of animal microRNAs. Nature, 431, 350–355.
Ason, B., Darnell, D. K., Wittbrodt, B. et al. (2007). Differences in vertebrate microRNA expression. (In press.)
Barad, O., Meiri, E., Avniel, al. (2004). MicroRNA detection by oligonucleotide microarrays: system establishment and expression profiling of human disease. Genome Research, 14, 2486–2494.
Bell, G. W., Yatskievych, T. A. and Antin, P. B. (2004). GEISHA, A whole-mount in situ hybridization gene expression screen in chicken embryos. Developmenta Dynamics, 229, 677–687.
Bentwich, I., Avniel, A., Karov, al. (2005). Identification of hundreds of conserved and nonconserved microRNAs. Nature Genetics, 37, 766–770.
Berezikov, E. and Plasterk, R. H. A. (2005). Camels and zebrafish, viruses and cancer: a microRNA update. Human Molecular Genetics, 14, R183–R190.
Brennecke, J., Hipfner, D. R., Stark, A., Russel, R. B. and Cohen, S. M. (2003). Bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell, 113, 25–36.
Calin, G. A., Sevignani, C., Dumitru, C. al. (2004). Human micro RNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proceedings of the National Academy of Sciences USA, 101, 2999–3004.
Chen, C. and Okayama, H. (1987). High efficiency transformation of mammalian cells by plasmid DNA. Molecular Cell Biology, 7, 2745–2752.
Darnell, D. K., Kaur, S., Stanislaw, S., Konieczka, J. K., Yatskievych, T. A. and Antin, P. B. (2007). MicroRNA expression during chick embryo development. (In press.)
Dostie, J., Mourelatos, Z., Yang, M., Sharma, A. and Dreyfus, G. (2003). Numerous microRNPs in neuronal cell containing novel microRNAs. RNA, 9, 180–186.
Duncan, L. M., Deeds, J., Hunter, al. (1998). Down-regulation of the novel gene melastatin correlates with potential for melanoma metastasis. Cancer Research, 58, 1515–1520.
Elbashir, S. M., Lendeckel, W. and Tuschl, T. (2001). RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes & Development, 15, 188–200.
Elmen, J., Zhang, H. Y., Zuber, al. (2004). Locked nucleic acid containing antisense oligonucleotides enhance inhibition of HIV-1 genome dimerization and inhibit virus replication. Federation of the European Biochemical Society Letters, 578, 285–290.
Elmen, J., Thonberg, H., Ljungberg, al. (2005). Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality. Nucleic Acids Research, 33, 439–447.
Giraldez, A. J., Cinalli, R. M., Glasner, M. al. (2005). MicroRNAs regulate brain morphogenesis in zebrafish. Science, 308, 833–838.
Griffiths-Jones, S. (2004). The microRNA registry. Nucleic Acids Research, 32, D109–D111.
Griffiths-Jones, S., Grocock, R. J., Dongen, S., Bateman, A. and Enright, A. J. (2006). miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Research, 34, D140–D144.
Hatfield, S. D., Scherbata, H. R., Fischer, K. al. (2005). Stem cell division is regulated by the microRNA pathway. Nature, 435, 974–978.
He, L. and Hannon, G. J. (2004). MicroRNAs: small RNAs with a big role in gene regulation. Nature Reviews in Genetics, 5, 522–532.
He, L., Thomson, J. M., Hemann, M. al. (2005). A microRNA polycistron as a potential human oncogene. Nature, 435, 828–833.
Hornstein, E., Mansfield, J. H., Yekta, al. (2005). The microRNA miR-196 acts upstream of Hoxb8 and Shh in limb development. Nature, 438, 671–674.
Johnson, S. M., Grosshans, H., Shingara, al. (2005). RAS is regulated by the let-7 microRNA family. Cell, 120, 635–647.
Kloosterman, W. P., Wienholds, E., Bruijn, E., Kauppinen, S. and Plasterk, R. H. A. (2006). In situ detection of miRNAs in animal embryos using LNA-modified oligonucteotide probes. Nature Methods, 3, 27–29.
Koshkin, A. A., Singh, S. K., Nielsen, al. (1998). LNA (Locked Nucleic Acids): synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine, and uracil bicyclonucleoside monomers, oligomerization, and unprecedented nucleic acid recognition. Tetrahedron, 54, 3607–3630.
Lancman, J. J., Caruccio, N. C., Harfe, B. al. (2005). Analysis of the regulation of lin-41 during chick and mouse limb development. Developmental Dynamics, 234, 948–960.
Lee, J. H., Koyano-Nakagawa, N., Naito, al. (1993). cAMP activates the IL-5 promoter synergistically with phorbol ester through the signaling pathway involving protein kinase A in mouse thyoma line El-4. Journal of Immunity, 151, 6135–6142.
Lee, Y., Jeon, K., Lee, J. T., Kim, S. and Kim, V. N. (2002). MicroRNA maturation: stepwise processing and subcellular localization. European Molecular Biology Organization Journal, 21, 4663–4670.
Legendre, M., Lambert, A. and Gautheret, D. (2005). Profile-based detection of microRNA precursors in animal genomes. Bioinformatics, 21, 841–845.
Lu, J., Getz, G., Miska, E. al. (2005). MicroRNA expression profiles classify human cancers. Nature, 435, 834–838.
Nieto, M. A., Patel, K. and Wilkinson, D. G. (1996). In situ hybridization analysis of chick embryos in whole mount and tissue sections. In Methods in Cell Biology, vol. 51. New York: Academic Press, Inc.
O'Donnell, K. A., Wentzel, E. A., Zeller, K. I., Dang, C. V. and Mendell, J. T. (2005). c-Myc-regulated microRNAs modulate E2F1 expression. Nature, 435, 839–843.
Ohler, U., Yekta, S., Lim, L. P., Bartel, D. P. and Burge, C. B. (2004). Patterns of flanking sequence conservation and a characteristic upstream motif for microRNA gene identification. RNA, 10, 1309–1322.
Pillai, R. S. (2005). MicroRNA function: multiple mechanisms for a tiny RNA?RNA, 11, 1753–1761.
Reinhart, B. J., Slack, F. J., Basson, al. (2000). The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature, 403, 901–906.
Rougvie, A. E. (2005). Intrinsic and extrinsic regulators of developmental timing: from miRNAs to nutritional cues. Development, 132, 3787–3798.
Stark, A., Brennecke, J., Bushati, N., Russell, R. B. and Cohen, S. M. (2005). Animal microRNAs confer robustness to gene expression and have a significant impact on 3′ UTR evolution. Cell, 123, 1133–1146.
Thomsen, R., Nielsen, P. S. and Jensen, T. H. (2005). Dramatically improved RNA in situ hybridization signals using LNA-modified probes. RNA, 11, 1745–1748.
Thomson, J. M., Parker, J., Perou, C. M. and Hammond, S. M. (2004). A custom microarray platform for analysis of microRNA gene expression. Nature Methods, 1, 47–53.
Tuschl, T., Zamore, P. D., Lehmann, R., Bartel, D. P. and Sharp, P. A. (1999). Targeted mRNAs degradation by double-stranded RNA in vitro. Genes & Development, 13, 3191–3197.
Valencia-Sanchez, M. A., Liu, J. D., Hannon, G. J. and Parker, R. (2006). Control of translation and mRNA degradation by miRNAs and siRNAs. Genes & Development, 20, 515–524.
Valoczi, A., Hornyik, C., Varga, al. (2004). Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. Nucleic Acids Research, 32, e175.
Wahlestedt, C., Salmi, P., Good, al. (2000). Potent and nontoxic antisense oligonucleotides containing locked nucleic acids. Proceedings of the National Academy of Sciences USA, 97, 5633–5638.
Watanabe, T., Takeda, A., Mise, al. (2005). Stage specific expression of microRNAs during Xenopus development. Federation of the European Biochemical Society Letter, 579, 318–324.
Weinholds, E., Kloosterman, W. P., Miska, al. (2005). MicroRNA expression in zebrafish embryonic development. Science, 309, 310–311.
Wightman, B., Ha, I. and Ruvkun, G. (1993). Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell, 75, 855–862.
Xie, X., Lu, J., Kulbokas, E. al. (2005). Systematic discovery of regulatory motifs in human promoters and 3′ UTRs by comparison of several mammals. Nature, 434, 338–345.
Zhao, Y., Samal, E. and Srivastava, D. (2005). Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis. Nature, 436, 214–220.