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. 2014 Oct 15;28(20):2205–2218. doi: 10.1101/gad.245142.114

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© 2014 Cai et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 2. — Cooperation of Wnt4 and Rspo1 is critical for MaSC reconstitution in vivo. (A) Isolated mammary cells were infected with GFP and mCherry scramble control lentivirus and cultured in Matrigel for colony formation. The infected cells were isolated using fluorescence and transplanted into recipients. Outgrowths (in yellow) with normal morphology were detected in nulliparous virgins (10 wk post-transplantation). When harvesting late pregnant recipients, a dense ductal system ending in a cluster of alveoli was observed in live fluorescence as well as with carmine staining. (B) Mammary cells with sh-Rspo1 knockdown led to decreased side branch formation in virgins and alveologenesis defects during pregnancy. (C) Mammary cells with sh-Wnt4 knockdown generated normal mammary outgrowth in virgins and during pregnancy. (D) Mammary cells with sh-Wnt4 and sh-Rspo1 double knockdown were able to form a colony in vitro but failed to generate mammary outgrowths in vivo. (E) Quantification of primary branches and the numbers of second and third branches in reconstituted mammary glands. (***) P < 0.05. (F) Infected cells were transplanted by limiting dilution. Rspo1 knockdown led to a decreased number of stem cells. Wnt4 and Rspo1 double knockdown completely abolished stem cell regeneration in vivo. Results are combined from three independent experiments. Bars: in colonies, 20 μm; in the whole mount, 2 mm. (G) qPCR analysis of mammary outgrowth indicated the successful knockdown Rspo1 (in B) or Wnt4 (in C) in vivo. mRNA was normalized to GAPDH. (***) P < 0.01.