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Science 338 (6112): 1357-1360

Copyright © 2012 by the American Association for the Advancement of Science

Sexually Dimorphic BDNF Signaling Directs Sensory Innervation of the Mammary Gland

Yin Liu1, Michael Rutlin1,*, Siyi Huang1, Colleen A. Barrick2, Fan Wang3, Kevin R. Jones4, Lino Tessarollo2, and David D. Ginty1,{dagger}

1 The Solomon H. Snyder Department of Neuroscience, Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
2 Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute–Frederick, National Institutes of Health, Frederick, MD 21702, USA.
3 Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.
4 Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.


Figure 1
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Fig. 1. Androgen-dependent, male-specific axon pruning leads to sexually dimorphic mammary gland innervation before male gland regression. (A to F) Tuj1 (yellow) and TO-PRO-3 (blue, nucleic acid stain) staining of mammary gland sections of embryos at different stages. This staining was used in all subsequent mammary gland innervation analyses unless otherwise specified. (G) Quantification of mammary gland innervation. (H to O) Mammary gland innervation of embryos treated with vehicle, flutamide, or testosterone propionate (8 hours). The vehicle group shown in (H) and (I) is the control for flutamide treatments; controls for the testosterone treatments were similar (not shown). Each experiment was done at least three times. (N) and (O) are quantifications of the data shown in (H) to (M). All statistical analyses shown in this figure and all subsequent figures (except Fig. 2, E and F) were done using two-way ANOVA with a Bonferroni post hoc test. Shown are the means ± SEM, n ≥ 5 embryos for each bar, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant. Scale bar: 50 μm.

 

Figure 2
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Fig. 2. BDNF-TrkB signaling is required for sensory innervation of the female mammary gland at E13. (A) Whole-mount X-gal staining of a late E13 female BDNFLacZ/+ embryo (21). (Inset) Cross section of one mammary gland. Arrows: rudimentary mammary glands (five on each side of the body, glands nos. 1 and 5 are located behind the limbs). (B and B') Costaining of GFP, Tuj1, and TO-PRO-3 of a late E13 female TrkBGFP/+ mammary gland. (C to F) Mammary gland innervation of late E13 female wild-type (WT), BDNFlacZ/LacZ (null), TrkBf/+, and TrkBf/f; AvilCre/+embryos. [The AvilCre/+ mouse is a sensory neuron–specific Cre driver mouse line (22). Statistical analyses in (E) and (F) were done using Student’s t test.] (G to K) Mammary gland innervation of late E13 wild-type and TrkBF616A/F616A female embryos that had been exposed to either vehicle or 1NMPP1 for 5 hours. For each bar, n ≥ 3 embryos in (E), (F), and (K). Scale bar: 50 μm.

 

Figure 3
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Fig. 3. The truncated form of TrkB is expressed in E13 male mammary mesenchymal cells. (A and B) X-gal staining of late E13 female and male BDNFLacZ/+ embryos, respectively. (C to F) Mammary gland sections of late E13 male and female embryos were stained with TO-PRO-3 (blue) and, in addition, (C and D) with TrkBECD and Tuj1 antibodies or (E and F) with a truncated form–specific TrkB antibody (TrkB.T1). Staining observed in the female section in (E) is due to nonspecific antibody binding to blood cells. All experiments were done at least three times. Scale bar: 50 μm.

 

Figure 4
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Fig. 4. Androgen-dependent mammary mesenchymal TrkB expression controls formation of the sexually dimorphic pattern of mammary gland innervation before male gland regression. (A to F) Mammary gland sections of late E13 male and female embryos exposed to vehicle, flutamide, or testosterone propionate (8 hours) were stained with TrkBECD (red) and Tuj1 (green) antibodies, and TO-PRO-3 (blue). The same staining is shown in all subsequent images in this figure. (G to K) Mammary mesenchymal TrkB expression and mammary gland innervation of wild-type and TrkB.T1 mutant male and female embryos. (H) A male mammary gland with relatively low mesenchymal TrkB expression. (I) A male mammary gland with relatively high mesenchymal TrkB expression. (L) Quantification of mammary gland innervation of wild-type and TrkB.T1 mutant embryos at late E13 (n ≥ 7 embryos for each bar). (M) The amount of mammary gland innervation of TrkB.T1 mutant male embryos is inversely correlated with residual mesenchymal TrkB expression level. Relative fiber density is fiber density of individual mammary glands divided by the average fiber density of the corresponding female mammary glands at the same position along the anterior-posterior axis at late E13. Each data point represents one mammary gland from a TrkB.T1 mutant male embryo (65 glands from eight mice).

 


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