Abstract
We describe an automated high-throughput method to measure protein levels in single nuclei in blastoderm embryos of Drosophila melanogaster by means of immunofluorescence. The method consists of a chain of specific algorithms assembled into an image processing pipeline. This pipeline transforms a confocal scan of an embryo stained with fluorescently tagged antibodies into a text file. This text file contains a numerical identifier for each nucleus, the coordinates of its centroid, and the average concentrations of three proteins in that nucleus. The central algorithmic component of the method is the automatic identification of nuclei by edge detection with the use of watersheds as an error-correction step. This method provides high-throughput quantification at cellular resolution.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Azpiazu N, Frasch M (1993) tinman and bagpipe: two homeobox genes that determine cell fates in the dorsal mesoderm of Drosophila. Genes Dev 7:1325–1340
Bullock S, Stauber M, Prell A, Hughes JR, Ish-Horowicz D, Schmidt-Ott U (2004) Differential cytoplasmic mRNA localisation adjusts pair-rule transcription factor activity to cytoarchitecture in dipteran evolution. Development 131:4251–4261
Chakraborty A (1996) Feature and module integration for image segmentation. Ph.D. thesis, Yale University
Chawla MK, Lin G, Olson K, Vazdarjanova A, Burke SN, McNaughton BL, Worley PF, Guzowski JF, Roysam B, Barnes CA (2004) 3D-catFISH: a system for automated quantitative three-dimensional compartmental analysis of temporal gene transcription activity imaged by fluorescence in situ hybridization. J Neurosci Methods 139:13–24
Crimmins TR (1985) Geometric filter for speckle reduction. Appl Opt 24:1438–1443
Driever W, Nüsslein-Volhard C (1988) A gradient of Bicoid protein in Drosophila embryos. Cell 54:83–93
Fleming MG (1996) Design of a high resolution image cytometer with open software architecture. Anal Cell Pathol 10:1–11
Gonzalez RC, Woods RE (2002) Digital image processing, 2nd edn. Prentice-Hall, Upper Saddle River
Harlow E, Lane D (1999) Tagging proteins. In: Cuddihy J, Kuhlman T, Barker P (eds) Using antibodies. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, pp 101–149
Hewitt GF, Strunk B, Margulies C, Priputin T, Wang XD, Amey R, Pabst B, Kosman D, Reinitz J, Arnosti DN (1999) Transcriptional repression by the Drosophila Giant protein: cis element positioning provides an alternative means of interpreting an effector gradient. Development 126:1201–1210
Houchmandzadeh B, Wieschaus E, Leibler S (2002) Establishment of developmental precision and proportions in the early Drosophila embryo. Nature 415:798–802
Hu M-K (1962) Visual pattern recognition by moment invariants. IRE Trans Inf Theory IT-8:179–187
Jaeger J, Blagov M, Kosman D, Kozlov KN, Manu, Myasnikova E, Surkova S, Vanario-Alonso CE, Samsonova M, Sharp DH, Reinitz J (2004a) Dynamical analysis of regulatory interactions in the gap gene system of Drosophila melanogaster. Genetics 167:1721–1737
Jaeger J, Surkova S, Blagov M, Janssens H, Kosman D, Kozlov KN, Manu, Myasnikova E, Vanario-Alonso CE, Samsonova M, Sharp DH, Reinitz J (2004b) Dynamic control of positional information in the early Drosophila embryo. Nature 430:368–371
Knowles DW, Keranen S, Biggin MD, Sudar D (2002) Mapping organism expression levels at cellular resolution in developing Drosophila. In: Three-dimensional and multidimensional microscopy: image acquisition and processing IX. SPIE Proceedings, vol 4621, pp 57–64
Kosman D, Reinitz J, Sharp DH (1997) Automated assay of gene expression at cellular resolution. In: Proceedings of the 1998 Pacific Symposium on Biocomputing. PSB Proceedings, pp 6–17
Kosman D, Small S, Reinitz J (1998) Rapid preparation of a panel of polyclonal antibodies to Drosophila segmentation proteins. Dev Genes Evol 208:290–294
Lawrence PA (1992) The making of a fly. Blackwell, Oxford
Mjolsness E, Sharp DH, Reinitz J (1991) A connectionist model of development. J Theor Biol 152:429–453
Myasnikova E, Samsonova A, Kozlov K, Samsonova M, Reinitz J (2001) Registration of the expression patterns of Drosophila segmentation genes by two independent methods. Bioinformatics 17:3–12
Myasnikova E, Samsonova M, Kosman D, Reinitz J (2005) Removal of background signal from in situ data on the expression of segmentation genes in Drosophila. Dev Genes Evol DOI: 10.1007/s00427-005-0472-2
Ortiz de Solórzano C, Garcia Rodriguez E, Jones A, Pinkel D, Gray JW, Sudar D, Lockett SJ (1999) Segmentation of confocal microscope images of cell nuclei in thick tissue sections. J Microsc 193:212–226
Ortiz de Solórzano C, Malladi R, Lelièvre SA, Lockett SJ (2001) Segmentation of nuclei and cells using membrane related protein markers. J Microsc 201:404–415
Poustelnikova E, Pisarev A, Blagov M, Samsonova M, Reinitz J (2004) A database for management of gene expression data in situ. Bioinformatics 20:2212–2221
Rasure J, Young M (1992) Open environment for image processing and software development. In: Image Processing and Interchange, SPIE Proceedings, vol 1659, pp 300–310
Reinitz J, Hou S, Sharp DH (2003) Transcriptional control in Drosophila. Complexus 1:54–64
Shen J, Castan S (1986) An optimal linear operator for edge detection. In: Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition, Miami, FL, pp 109–114
Spirov AV, Holloway D (2003) Making the body plan: precision in the genetic hierarchy of Drosophila embryo segmentation. In Silico Biol 3:89–100
Tomancak P, Beaton A, Weiszmann R, Kwan E, Shu S, Lewis SE, Richards S, Ashburner M, Hartenstein V, Celniker SE, Rubin GM (2002) Systematic determination of patterns of gene expression during Drosophila embryogenesis. Genome Biol 3(12):RESEARCH0088
Umesh Adiga PS, Chaudhuri BB (1999) Efficient cell segmentation tool for confocal microscopy tissue images and quantitative evaluation of FISH signal. Microsc Res Tech 44:49–68
Vincent L (1993) Morphological grayscale reconstruction in image analysis: applications and efficient algorithms. In IEEE Transactions Image on Processing volume 2, pp 176–201
Wu X, Vasisht V, Kosman D, Reinitz J, Small S (2001) Thoracic patterning by the Drosophila gap gene hunchback. Dev Biol 237:79–92
Acknowledgements
We thank Konstantin Kozlov for comments on the manuscript. This work was supported financially by the National Institutes of Health.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by S. Roth
H. Janssens and D. Kosman contributed equally to this paper.
Electronic Supplementary Material
Rights and permissions
About this article
Cite this article
Janssens, H., Kosman, D., Vanario-Alonso, C.E. et al. A high-throughput method for quantifying gene expression data from early Drosophila embryos. Dev Genes Evol 215, 374–381 (2005). https://doi.org/10.1007/s00427-005-0484-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00427-005-0484-y