Ectodermal influx and cell hypertrophy provide early growth for all murine mammary rudiments, and are differentially regulated among them by Gli3 - PubMed (original) (raw)

doi: 10.1371/journal.pone.0026242. Epub 2011 Oct 27.

Victor Racine, Peter Jagadpramana, Li Sun, Weimiao Yu, Tiehua Du, Bradley Spencer-Dene, Nicole Rubin, Lendy Le, Delphine Ndiaye, Saverio Bellusci, Klaus Kratochwil, Jacqueline M Veltmaat

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Ectodermal influx and cell hypertrophy provide early growth for all murine mammary rudiments, and are differentially regulated among them by Gli3

May Yin Lee et al. PLoS One. 2011.

Abstract

Mammary gland development starts in utero with one or several pairs of mammary rudiments (MRs) budding from the surface ectodermal component of the mammalian embryonic skin. Mice develop five pairs, numbered MR1 to MR5 from pectoral to inguinal position. We have previously shown that Gli3(Xt-J/Xt-J) mutant embryos, which lack the transcription factor Gli3, do not form MR3 and MR5. We show here that two days after the MRs emerge, Gli3(Xt-J/Xt-J) MR1 is 20% smaller, and Gli3(Xt-J/Xt-J) MR2 and MR4 are 50% smaller than their wild type (wt) counterparts. Moreover, while wt MRs sink into the underlying dermis, Gli3(Xt-J/Xt-J) MR4 and MR2 protrude outwardly, to different extents. To understand why each of these five pairs of functionally identical organs has its own, distinct response to the absence of Gli3, we determined which cellular mechanisms regulate growth of the individual MRs, and whether and how Gli3 regulates these mechanisms. We found a 5.5 to 10.7-fold lower cell proliferation rate in wt MRs compared to their adjacent surface ectoderm, indicating that MRs do not emerge or grow via locally enhanced cell proliferation. Cell-tracing experiments showed that surface ectodermal cells are recruited toward the positions where MRs emerge, and contribute to MR growth during at least two days. During the second day of MR development, peripheral cells within the MRs undergo hypertrophy, which also contributes to MR growth. Limited apoptotic cell death counterbalances MR growth. The relative contribution of each of these processes varies among the five MRs. Furthermore, each of these processes is impaired in the absence of Gli3, but to different extents in each MR. This differential involvement of Gli3 explains the variation in phenotype among Gli3(Xt-J/Xt-J) MRs, and may help to understand the variation in numbers and positions of mammary glands among mammals.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Relationship between Gli3 expression and mammary rudiment growth in mouse embryos.

A–H: Hematoxylin/Eosin stained central transversal sections through MRs. Black contours in B surround the MR (solid) and mammary mesenchyme (dotted). Scale-bar in A: 100 µm in A–H. I–J: Scanning electron micrographs showing the outlet of the prospective milk-canal in a wt MR2, and the outwardly protruding Gli3Xt-J/Xt-J MR2 at E15.5. K–N: Carmine-stained skins. Arrowheads indicate some end buds (white) and the nipple (black). O: Volumetric growth curve of MRs. P–U′: Bright-field images of wt MRs and corresponding dark-field images with the radio-active in situ hybridization signal of a Gli3 mRNA probe in white. V: Quantification of Gli3 expression in micro-array data of each MR, ectoderm (Ect) and mesenchyme (Mes). Dashed error-bars at E13.5 extend between n = 2 measurements.

Figure 2

Figure 2. Proliferative activity in wt and Gli3Xt-J/Xt-J MRs and adjacent tissues.

A–Y: Immunohistochemical detection of BrdU-incorporation (black) at 2 hours post-labeling, with hematoxylin-counterstain (blue). Dashed yellow lines outline the MR. Left is ventral ectoderm, in some panels indicated with *. Scale-bar in A: 100 µm in A–Y. Z-AI: Quantification of BrdU-incorporation in entire MRs and their adjacent tissues, represented in A–Y. Z: Quality control image of a wt E13.5 MR1 as generated with our software. Colored outlines demarcate dorsal ectoderm (d.ect., green), ventral ectoderm (v.ect., yellow), MR core (c, orange), MR periphery (p, red), MR neck (n, purple), mammary mesenchyme (m.m., turquoise), and dermal mesenchyme (d.m., blue). AA,AB: Relationship between MR-volume and BrdU-incorporation. AC–AE: The BrdU+ve fraction of all nuclear pixels in MRs and their adjacent ectoderm. AF–AH: The BrdU+ve fraction of all nuclear pixels in ‘pre’mammary or mammary and dermal mesenchyme (mes). AI: Differences in BrdU-incorporation between the ventral and dorsal ectoderm (fraction in dorsal side subtracted from fraction in ventral side per embryo). Asterisks denote a significantly difference (_p<_0.05) between both sides.

Figure 3

Figure 3. Apoptosis in MRs and their adjacent tissues is regulated by Gli3.

A–P: Epifluorescence images of TUNEL-stained (green) and DAPI-counterstained (blue) central transversal sections through MRs, outlined by dashed white lines. Red insets: magnification of the apical area of the MR. White insets: magnification of a TUNEL+ve region in the mammary mesenchyme. Left is ventral ectoderm, in some panels indicated with *. Scale-bar in A: 100 µm in A–P.

Figure 4

Figure 4. Ectodermal influx provides growth of all MRs and is perturbed for Gli3Xt-J/Xt-J MR2 and MR4.

A–P: Immunohistochemical detection of BrdU-incorporation (black) at 24 hours post-labeling, with hematoxylin-counterstain (blue). Panels F–J and F′–J′ show a random versus primarily proximal distribution of labeled cells observed in 60% and 40% of wt MRs respectively. Dashed yellow lines outline the MR. Left is ventral ectoderm, in some panels indicated with *. Scale-bar in A: 100 µm in A–P. Q–V: Quantification of BrdU-labeling in entire MRs and adjacent tissues represented in A–P, by absolute number of BrdU+ve pixels (Q) or BrdU+ve fraction of all nuclear pixels (R) in all segmented tissues combined; or BrdU+ve fraction of all nuclear pixels (S,T) or absolute number (U,V) of BrdU+ve pixels in the MRs.

Figure 5

Figure 5. Unique compartmentalization of BrdU+ve cells among individual MRs at E13.5.

A–F: BrdU+ve fraction of all nuclear pixels per MR (y-axis), subdivided proportionally by distribution over core, periphery, and neck if present. G: 3D-reconstruction of MRs based on serial sections stained for BrdU as in Figs. 2 and 5. Ectoderm/epidermis in green, with black BrdU+ve nuclei; MR-epithelium in red , with blue BrdU+ve nuclei.

Figure 6

Figure 6. A model of the role of Gli3 in regulating mammary rudiment induction and early growth.

See discussion for explanation. Thickness of arrows relates to the relative extent of involvement of Gli3 in a process.

References

    1. Veltmaat JM, Van Veelen W, Thiery JP, Bellusci S. Identification of the mammary line in mouse by Wnt10b expression. Dev Dyn. 2004;229:349–356. - PubMed
    1. Veltmaat JM, Mailleux AA, Thiery JP, Bellusci S. Mouse embryonic mammogenesis as a model for the molecular regulation of pattern formation. Differentiation. 2003;71:1–17. - PubMed
    1. Cunha GR, Young P, Christov K, Guzman R, Nandi S, et al. Mammary phenotypic expression induced in epidermal cells by embryonic mammary mesenchyme. Acta Anat (Basel) 1995;152:195–204. - PubMed
    1. Balinsky BI. On the prenatal growth of the mammary gland rudiment in the mouse. J Anat. 1950;84:227–235. - PMC - PubMed
    1. Balinsky BI. On the Developmental Processes in Mammary Glands and other Epidermal Structures. Transactions of the Royal Society of Edinburgh. 1949–1950;62:1–31.

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