Pups generated from self-crosses of <i>Spry2</i> heterozygous mice.

<p>Pups were genotyped on postnatal day 1 (P1; n = 42) and upon weaning on postnatal day 21 (P21; n = 359). Note the actual frequencies (Act.) of both <i>Spry2</i>^Δ/+ and <i>Spry2</i>^+/+ were more than the expected frequencies (Exp.) because a portion of the <i>Spry2</i>^Δ/Δ pups died prior to weaning.</p

Modulation of Fibroblast Growth Factor Signaling Is Essential for Mammary Epithelial Morphogenesis

<div><p>Fibroblast growth factor (FGF) signaling is essential for vertebrate organogenesis, including mammary gland development. The mechanism whereby FGF signaling is regulated in the mammary gland, however, has remained unknown. Using a combination of mouse genetics and 3D ex vivo models, we tested the hypothesis that <i>Spry2</i> gene, which encodes an inhibitor of signaling via receptor tyrosine kinases (RTKs) in certain contexts, regulates FGF signaling during mammary branching. We found that <i>Spry2</i> is expressed at various stages of the developing mammary gland. Targeted removal of <i>Spry2</i> function from mammary epithelium leads to accelerated epithelial invasion. <i>Spry2</i> is up-regulated by FGF signaling activities and its loss sensitizes mammary epithelium to FGF stimulation, as indicated by increased expression of FGF target genes and epithelia invasion. By contrast, <i>Spry2</i> gain-of-function in the mammary epithelium results in reduced FGF signaling, epithelial invasion, and stunted branching. Furthermore, reduction of <i>Spry2</i> expression is correlated with tumor progression in the MMTV-PyMT mouse model. Together, the data show that FGF signaling modulation by <i>Spry2</i> is essential for epithelial morphogenesis in the mammary gland and it functions to protect the epithelium against tumorigenesis.</p></div

Gain of <i>Spry2</i> function in the mammary epithelium causes retarded epithelial branching.

<p>(<b>A</b>) Schematic diagram depicting the <i>Spry2</i>-GOF transgene. The β<i>-Geo</i> gene was driven by the CAGG promoter and followed by a triple poly-adenylation sequence (3x pA). Upon Cre-mediated recombination, the β<i>-Geo</i> gene was deleted and the mouse <i>Spry2</i> and human placental alkaline phosphatase (PLAP), constructed as a bi-cistronic mRNA containing an internal ribosome entry site (IRES) directing PLAP translation, were expressed. (<b>B–C</b>) Assay for PLAP activities in control (M-Cre or <i>Spry2</i>-GOF) and mutant (M-Cre;<i>Spry2</i>-GOF) #3 glands from adult female mice at 15-weeks of age. Note PLAP activities were detected in mutant (<b>C</b>) but not in control glands (<b>B</b>). The area in the dashed red box is highlighted in a close-up picture in the inset, illustrating the branching network that was positive for PLAP activities. (<b>D–I</b>) The mammary branching tree from #4 glands at the postnatal stages indicated. Samples were assayed for PLAP activities and were then stained with Carmine Red. (<b>D–F</b>) glands from control mice; (<b>G–I</b>) glands from mutant mice. Arrows indicate the extent of ductal penetration in the fat pad. Dotted white line illustrates the epithelial invasion front. Insets in (<b>G</b>), (<b>H</b>), and (<b>I</b>) show high-magnification views of the rudimentary ductal tree (area in dashed box), illustrating only some of the mammary epithelial cells showed PLAP activities due to the mosaic activity of the M-Cre transgene. Solid arrowheads indicate TEBs from 4-week (<b>G</b>) and 7-week (<b>H</b>) mammary glands that were more heavily stained for PLAP activities than other TEBs indicated by open arrowheads (<b>H</b>). These data suggest that the mammary glands from the bi-transgenic mice (M-Cre;<i>Spry2</i>-GOF) are mosaic, containing both Cre-expressing and non-Cre-expressing cells. (<b>J</b>, <b>K</b>) Quantitative comparisons of ductal penetration and branch point formation between control and mutant glands. At 7 weeks, ductal penetration measurements were 7.0±1.9 (control, n = 6) and 1.8±1.0 (mutant, n = 6); at 8 weeks, the measurements were 9.9±1.2 (control, n = 4) and 7.7±2.3 (mutant, n = 10); at 10 weeks, they were 12.4±0.3 (control, n = 8) and 12.7±0.04 (mutant, n = 4). Measurements of branching points were 2.1±0.3 (control) and 1.2±0.2 (mutant) at 7 weeks, 1.7±0.1 (control) and 2.3±0.4 (mutant) at 8 weeks, and 2.3±0.2 (control) and 2.4±0.2 (mutant) at 10 weeks. Values shown are the mean ± SD for each data point: *, P<0.05, unpaired, two-tailed Student’s <i>t</i> test. Scale bars: 2.5 mm. N is the number of mammary glands examined.</p

<i>Spry2</i> null mice show stunted epithelial branching due to malnourishment.

<p>(<b>A–C</b>) <i>Spry2</i> mRNA expression as detected by quantitative RT-PCR (qPCR). (<b>A</b>) <i>Spry2</i> mRNA expression was measured by qPCR using RNA harvested from mammary glands from female mice at 3-weeks, 5-weeks, and 10-weeks of age as virgins, during pregnancy (P) on day 5 and 17, on day 1 of lactation (Lac), and on day 1, 3, and 10 of involution (Inv). <i>Spry2</i> expression at 3-weeks was set as base value against which other stages were compared. Abbreviations: wks, weeks; P, pregnancy; L, lactation; Inv, involution. (<b>B</b>, <b>C</b>) MECs were sorted based on their expression of CD24 and Integrin-α6 (CD49f). CD24^medCD49f^hi cells were basal (ba), whereas CD24^hiCD49f^l°^w cells and CD24^l°^wCD49f^l°^w were luminal (lu) and stromal (st), respectively. RNA was harvested from the three cell partitions to generate DNA templates for qPCR reactions (<b>C</b>). (<b>D</b>, <b>E</b>) The mammary branching tree at 6-weeks of age, as revealed by Carmine Red staining of glands in wholemount. Proximal (close to the nipple) is to the left and distal is to the right. Arrowheads indicate TEBs at the tips of invading mammary epithelium, which persist until branching morphogenesis ceases in adult glands. Arrows indicate the extent of ductal penetration in the fat pad. Note epithelial branching was severely stunted in (<b>E</b>) mutant (<i>Spry2</i>^Δ/Δ; n = 8) mice when compared with (<b>D</b>) control (<i>Spry2</i>^Δ/+; n = 12) mice. Scale bars: 2 mm. Abbreviation: epi, epithelium; st, stroma; LN, lymph node. (<b>F–M</b>) <i>Spry2</i> null mice showed growth retardation (<b>F</b>, <b>G</b>) and an insufficiency in energy storage (<b>H–M</b>). (<b>F</b>) Growth curve of pups born from <i>Spry2</i>^Δ/+ crosses. Weights between <i>Spry2</i>^Δ/+ (n = 15) and <i>Spry2</i>^+/+ (n = 4) mice were indistinguishable and combined. Values shown are the mean ± SD for each data point. (<b>G</b>) Dorsal view of typical appearances of <i>Spry2</i>^Δ/+ and <i>Spry2</i>^Δ/Δ mice at 12-weeks of age. Note <i>Spry2</i>^Δ/Δ mice were shorter than normal and had enlarged midsection (flanked by dotted black lines) due to distended intestines (not shown). Scale bars: 2 cm. (<b>H–M</b>) Glycogen and lipid storage, as revealed by Periodic Acid-Schiff and Oil-Red-O staining, respectively, and histology of white adipose tissue from <i>Spry2</i>^Δ/+ (<b>H</b>–<b>J</b>) and <i>Spry2</i>^Δ/Δ mice (<b>K</b>–<b>M</b>). Note that <i>Spry2</i>^Δ/Δ mutant liver lacked glycogen (<b>K</b>) and lipid storage (<b>L</b>) as was evident in control liver (purple-magenta color in <b>H</b> and red droplets in <b>I</b>); moreover, adipocytes from white adipose tissue in <i>Spry2</i>^Δ/Δ mutant (<b>M</b>) mice were smaller than normal (<b>J</b>). Scale bars: 100 μm.</p

<i>Spry2</i> null epithelium shows enhanced FGF signaling activities and increased epithelial branching activities.

<p>(<b>A</b>) Expression, as measured by qPCR, of <i>Spry2</i> and target genes of FGF signaling, including <i>Etv4</i>, <i>Etv5</i>, and <i>Mkp3</i>, in response to a 24-hour treatment of FGF2 (10 nM) or FGF10 (10 nM). Expression is relative to that of the untreated samples. Values shown are the mean ± standard deviation (SD) of three independent experiments. Statistically significant differences of p<0.05 (t test) were observed between expression of untreated and treated samples for all genes except for <i>Etv5</i> in response to FGF2 and FGF10 treatment. (<b>B</b>) Schematic diagram depicting the experimental procedure in sample preparation, treatment, and analysis. Mammary organoids were prepared from <i>Spry2</i>^+/+ and <i>Spry2</i>^fl/fl mice and were infected with adenovirus-Cre-GFP, which generated control (<i>Spry2</i>^+/+) and mutant (<i>Spry2</i>^Δ/Δ) organoids, respectively. Transduced cells were then purified by FACS based on their expression of GFP before they were subjected to analyses on gene expression and epithelial morphogenesis in the presence or absence of FGF2 or FGF10. (<b>C–D</b>) Expression, as measured by qPCR, of <i>Etv4</i>, <i>Etv5</i>, and <i>Mkp3</i> in control and mutant MECs in response to 24-hour treatment of FGF2 (200 ng/ml, <b>C</b>) or FGF10 (200 ng/ml, <b>D</b>). Expression is relative to that of the control samples. Statistically significant differences of p<0.05 (t test) were observed between expression of control and mutant samples for all genes except for <i>Etv5</i> in response to FGF2 treatment and <i>Etv4</i> in response to FGF10 treatment. (<b>E–I</b>) in vitro branching assay in which control (<b>E</b>, <b>F</b>) and mutant organoids (<b>G</b>, <b>H</b>) were subjected to cultures in basal medium with (<b>F</b>, <b>H</b>) or without FGF2 (<b>E</b>, <b>G</b>). When stimulated by FGF2 at progressively higher concentrations from 0.025 nM to 0.5 nM, a progressively higher percentage of organoids underwent branching. At 1.0 nM and 2.5 nM, FGF2 did not stimulate a higher percentage of branched organoids to form. In addition to their differences in branching kinetics, <i>Spry2</i>^Δ/Δ organoids overall formed larger branched structures than control organoids. Scale bars: 100 μm. (<b>I</b>) Quantitative comparisons of control and mutant MECs in their ability to undergo epithelial branching in vitro. Data were from experiments repeated three times or more. At least 100–150 organoids were examined for each treatment conditions. Values shown are the mean ± SD for each data point: *P<0.0005, unpaired, two-tailed Student’s <i>t</i> tests.</p