In situ hybridization with 3DNA™ dendrimers is a novel tool for detecting low levels of mRNA in tissue sections and whole embryos. Fluorescently labeled dendrimers were used to identify cells that express mRNA for the skeletal muscle transcription factor MyoD in the early chick embryo. A small population of MyoD mRNA positive cells was found in the epiblast prior to the initiation of gastrulation, two days earlier than previously detected using enzymatic or radiolabeled probes for mRNA. When isolated from the epiblast and placed in culture, the MyoD mRNA positive cells were able to differentiate into skeletal muscle cells. These results demonstrate that DNA dendrimers are sensitive and precise tools for identifying low levels of mRNA in single cells and tissues.
George-Weinstein M, Gerhart JV, Reed R, Flynn J, Callihan B, Mattiacci M, Miehle M, Foti G, Lash JW, Weintraub H. Skeletal myogenesis: the preferred pathway of chick embryo epiblast cells in vitro. Dev Biol 1996; 173:279–291.
Sassoon D, Lyon G, Wright WE, Lin V, Lassar A, Weintraub H, Buckingham M. Expression of two myogenic regulatory factors myogenin and MyoD1 during mouse embryogenesis. Nature 1989; 341:303–307.
Pownall ME, Emerson CP. Sequential activation of three myogenic regulatory genes during somite morphogenesis in quail embryos. Dev Biol 1992; 151:67–79.
Charles de la Brousse F, Emerson CP. Localized expression of a myogenic regulatory gene, qmf1 in the somite dermatome of avian embryos. Genes Dev 1990; 4:567–581.
Gerhart JV, Baytion M, DeLuca S, Getts R, Lopez C, Niewenhuis R, Nilsen TW, Olex S, Weintraub H, George-Weinstein M. DNA dendrimers localize MyoD mRNA in presomitic tissues of the chick embryo. J Cell Biol 2000; 149:825–833.
Nilsen T, Grayzel J, Prensky W. Dendritic nucleic acid structures. J Theor Biol 1997; 187:273–284.
Volgelbacker HH, Getts RC, Tian N, Labaczewski R, Nilsen TW. DNA dendrimers: assembly and signal amplification. Proc Am Chem Soc 1997; 76:458–460.
Wang J, Jiang M, Nilsen TW, Getts RC. Dendritic nucleic acid probes for DNA biosensors. J Am Chem Soc 1998; 120:8281–8282.
Gerhart JV, Neely C, Stewart B, Perlman J, Beckman D, Wallon M, Knudsen K, George-Weinstein M. Epiblast cells that express MyoD recruit plutipotent cells to the skeletal muscle lineage. J Cell Biol 2004; 164:739–746.
Dechesne CA, Wei J, Eldridge L, Gannoun-Zaki P, Millasseau L, Bougueleret D, Caterina D, Paterson BM. Ebox and MEF-2 independent muscle-specific expression, positive autoregulation, and cross-activation of the chicken MyoD (CMD1) promoter reveal an indirect regulatory pathway. Mol Cell Biol 1994; 14:869–877.
Dugaiczyk A, Haron JR, Stone EM, Dennison OE, Rothblum KN, Schwartz RJ. Cloning and sequencing of a deoxyribonucleic acid copy of a glyceraldehydes-3-phosphate dehydrogenase messenger ribonucleic acid isolated from chicken muscle. Biochemistry 1983; 22:1605–1613.
Freyer GC, Robbins J. The analysis of a chicken myosin heavy chain cDNA clone. J Biol Chem 1983; 258:7149–7154.
Sassoon D, Rosenthal N. Detection of messenger RNA by in situ hybridization. Methods Enzymol 1993; 225:384–404.
Raap KA, van de Rijke FM, Dirks RW. MRNA in situ hybridization to in vitro cultured cells. In: Choo KHA editor. Method in Molecular Biology. Vol 33. Totowa, NJ: Humana Press; 1994. p.293–300.
Hamburger V, Hamilton HL. A series of normal stages in development of the chick embryo. J Morphol 1951; 88:49–92.
Gerhart JV, Bast B, Neely C, Iem S, Amegbe P, Niewenhuis R, Miklasz S, Cheng PF, George-Weinstein M. MyoD-positive myoblast are present in mature fetal organs lacking skeletal muscle. J Cell Biol 2001; 155:381–391.
George-Weinstein M, Gerhart JV, Foti G, Lash JW. Maturation of myogenic and chondrogenic cells in the presomitic mesoderm of the chick embryo. Exp Cell Res 1994; 211:263–274.
Kitner CR, Brockes JP. Monoclonal antibodies identify blastemal cells derived from dedifferentiating muscle in newt limb regeneration. Nature 1984; 308:67–69.
Ott MO, Bober E, Lyons G, Arnold H, Buckingham M. Early expression of the myogenic regulatory gene, myf-5 in precursor cells of skeletal muscle in the mouse embryo. Development 1991; 111:1097–1107.
Tajbakhsh S, Vivarelli E, Cusella-De Angelis G, Rocancourt D, Buckingham M, Cossu G. A population of myogenic cells derived from the mouse neural tube. Neuron 1994; 13:813–821.
Bader D, Masakki T, Fischman DA. Immunochemical analysis of myosin heavy chain during avian embryogenesis in vivo and in vitro. J Cell Biol 1995; 95:763–770.
DiLullo C, Gerhart JV, George-Weinstein M. Preparation of chick-striated muscle cultures. In: Tuan R and Lo C, Editors, Methods in Molecular Biology, Humana Press, Totowa, NJ. 2000; 137: 337–349.
Published: July 16, 2004.
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Gerhart, J., Baytion, M., Perlman, J. et al. Visualizing the needle in the haystack: In situ hybridization with fluorescent dendrimers. Biol. Proced. Online 6, 149–156 (2004). https://doi.org/10.1251/bpo84
- MyoD protein
- In situ hybridization