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Figure 4. Expression of human OSR1 in animal caps. Animal caps derived from embryos injected with OSR1 mRNA were cultured in Steinberg's solution, retinoic acid (RA, 10-4M) and/or activin A (ActA, 10 ng/ml) as indicated. To avoid a too high lethality only 150 pg mRNA was injected. (A) Classification of whole-mount immunostained animal caps used for the analysis of pronephric tissue induction. The immunostaining was done with a mixture of the antibodies 3G8 and 4A6: - no pronephric tissue, + two or more pronephric cell patches or a small area of pronephric cells, +++ pronephric tubule like structures. Bar = 200 μm (B) Exogenous OSR1 is expressed in injected embryos. After cutting the animal caps protein extracts of the remaining late blastula stage embryos injected with OSR1 mRNA were used for Western blot analysis with myc-tag specific antibody. Lane 1 and 2 refer to two pools of ten embryos. The exogenous myc-OSR1 is marked by an arrow. (C) The pronephros induction potential in animal caps was assayed after four days using the categories given in panel A. N refers to the number of animal caps investigated. (D) Quantitative RT-PCR analysis for the expression of early nephrogenic transcription factors (TFs) of animal caps after three hours and treated as indicated in (C). The bar represents the values of two independent animal cap pools (N = 30) tested. The results of OSR1 injection with retinoic acid treatment alone or in combination with activin A are each from one animal cap pool. Data obtained for hnf1b expression were only from one pool tested except for OSR1 injection alone. Image published in: Drews C et al. (2011) Copyright ©2011 Drews et al; licensee BioMed Central Ltd. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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