Stromal Gli2 activity coordinates a niche signaling program for mammary epithelial stem cells

Double duty for mammary stem cell niche The stem cell niche is a complex local signaling microenvironment that regulates stem cell activity for tissue and organ maintenance and regeneration. As well as responding locally, during puberty, the mammary gland stem cell niche also responds to systemic hormonal signals. Zhao et al. have found that Gli2, a transcriptional effector of Hedgehog signaling, coordinates the niche-signaling program and activates expression of receptors for the mammatrophic hormones estrogen and growth hormone throughout the mammary gland (see the Perspective by Robertson). Disease may result not only from stem cell defects, but also from dysregulation of the microenvironment. Science, this issue p. eaal3485; see also p. 250 The GLI2 transcription factor regulates stromal cell expression of signals controlling breast epithelial stem cell activity. INTRODUCTION The stem cell niche is a complex signaling microenvironment that acts locally to sustain stem cell activity in tissue maintenance and regeneration. Although the cellular constitution and signaling activity of the stem cell niche is coming into focus in a variety of tissues, genetic regulatory factors that specify the niche are less clear. The activity of such factors is particularly intriguing in organs such as the breast, where the niche provides local signals for tissue homeostasis but also must be entrained by circulating hormones that induce the dramatic changes of puberty. RATIONALE The epithelial stem cells of a variety of organs respond to secreted signals generated in subjacent stromal cells, in a manner often dependent on activity of the Hedgehog (Hh) signaling pathway. Gli2, which encodes the major transcriptional effector of Hh signaling, is expressed in a subset of stromal cells adjacent to the ductal epithelium of the mouse mammary gland and is highly expressed at terminal end buds and end structures of the pubertal and adult mammary gland, respectively, which represent the sites of mammary epithelial stem cells. RESULTS Stromal ablation of a conditional Gli2fl allele with Fsp1Cre, a stromally expressed recombinase allele (producing Gli2∆S mice), caused a delay in mammary ductal development, reduced the number of mammary gland stromal cells and volume of extracellular matrix, and caused abnormal mammary duct distension. Stromal Gli2 ablation did not alter development of the ovary or pituitary, nor their production of mammatrophic hormones such as estrogen or growth hormone, but did affect ductal regeneration, as indicated by a fivefold decrease in outgrowth efficiency of mammary stem cells (MaSCs) transplanted into Gli2∆S mammary glands. These findings suggest that Gli2 specifies a stromal niche signaling program that critically regulates MaSC activity. FACS-isolated mammary stromal cells showed Gli2-dependent expression of factors that stimulate epithelial stem cell renewal, ductal outgrowth, and morphogenesis, including specific members of the IGF, WNT, FGF, and HGF families of secreted peptides. Single-cell analysis showed expression of these factors in a subset of stromal cells in a manner dependent on Gli2 function. Receptors for the mammatrophic hormones estrogen and growth hormone were also expressed in a Gli2-dependent manner, and estrogen and growth hormone treatment of cultured stromal cells induced these secreted peptide factors, suggesting that a critical role of Gli2 in mammary stromal niche cells is to render these cells responsive to systemic mammatrophic hormones. Supporting this conclusion, mammary gland implants of polymer fragments releasing IGF1 and WNT2 rescued Gli2∆S ductal growth phenotypes, whereas growth hormone–releasing polymer did not. CONCLUSION We find that the Hedgehog pathway transcriptional effector GLI2 specifies a stromal cell niche signaling program that supports mammary epithelial stem cells in pubertal and virgin adult mice. This program includes expression of factors such as IGF and WNT, and GLI2 acts in part by causing expression of receptors for hormones such as estrogen and growth hormone, thus entraining local stem cell niche activity to systemic mammatrophic hormones. Our work illustrates the central role of the stromal niche in controlling epithelial stem cell activity and suggests that niche failure may underlie pathogenesis of certain diseases, including the deficient breast development and hormonal insensitivity associated with the human disorder, combined pituitary hormone deficiency. Stromal GLI2 coordinates mammary stem cell niche signaling program. Transcriptional regulation by Hedgehog pathway effector GLI2 in stromal cells of the mammary gland coordinates a hormone-responsive niche signaling program that directs epithelial stem cell activity during the changes of puberty (schematic, left). Gli2-deficient stromal cells in mouse mammary are unable to support normal ductal outgrowth and morphogenesis of transplanted, green fluorescent protein–labeled stem cells (right). The stem cell niche is a complex local signaling microenvironment that sustains stem cell activity during organ maintenanceorgan maintenance and regeneration. The mammary gland niche must support its associated stem cells while also responding to systemic hormonal regulation that triggers pubertal changes. We find that Gli2, the major Hedgehog pathway transcriptional effector, acts within mouse mammary stromal cells to direct a hormone-responsive niche signaling program by activating expression of factors that regulate epithelial stem cells as well as receptors for the mammatrophic hormones estrogen and growth hormone. Whereas prior studies implicate stem cell defects in human disease, this work shows that niche dysfunction may also cause disease, with possible relevance for human disorders and in particular the breast growth pathogenesis associated with combined pituitary hormone deficiency.