Cryptic Patterning of Avian Skin Confers a Developmental Facility for Loss of Neck Feathering

Vertebrate skin is characterized by its patterned array of appendages, whether feathers, hairs, or scales. In avian skin the distribution of feathers occurs on two distinct spatial levels. Grouping of feathers within discrete tracts, with bare skin lying between the tracts, is termed the macropattern, while the smaller scale periodic spacing between individual feathers is referred to as the micropattern. The degree of integration between the patterning mechanisms that operate on these two scales during development and the mechanisms underlying the remarkable evolvability of skin macropatterns are unknown. A striking example of macropattern variation is the convergent loss of neck feathering in multiple species, a trait associated with heat tolerance in both wild and domestic birds. In chicken, a mutation called Naked neck is characterized by a reduction of body feathering and completely bare neck. Here we perform genetic fine mapping of the causative region and identify a large insertion associated with the Naked neck trait. A strong candidate gene in the critical interval, BMP12/GDF7, displays markedly elevated expression in Naked neck embryonic skin due to a cis-regulatory effect of the causative mutation. BMP family members inhibit embryonic feather formation by acting in a reaction-diffusion mechanism, and we find that selective production of retinoic acid by neck skin potentiates BMP signaling, making neck skin more sensitive than body skin to suppression of feather development. This selective production of retinoic acid by neck skin constitutes a cryptic pattern as its effects on feathering are not revealed until gross BMP levels are altered. This developmental modularity of neck and body skin allows simple quantitative changes in BMP levels to produce a sparsely feathered or bare neck while maintaining robust feather patterning on the body

Krüppel-like Transcription Factor KLF10 Suppresses TGFβ-Induced Epithelial-to-Mesenchymal Transition via a Negative Feedback Mechanism

Abstract TGFβ–SMAD signaling exerts a contextual effect that suppresses malignant growth early in epithelial tumorigenesis but promotes metastasis at later stages. Longstanding challenges in resolving this functional dichotomy may uncover new strategies to treat advanced carcinomas. The Krüppel-like transcription factor, KLF10, is a pivotal effector of TGFβ/SMAD signaling that mediates antiproliferative effects of TGFβ. In this study, we show how KLF10 opposes the prometastatic effects of TGFβ by limiting its ability to induce epithelial-to-mesenchymal transition (EMT). KLF10 depletion accentuated induction of EMT as assessed by multiple metrics. KLF10 occupied GC-rich sequences in the promoter region of the EMT-promoting transcription factor SLUG/SNAI2, repressing its transcription by recruiting HDAC1 and licensing the removal of activating histone acetylation marks. In clinical specimens of lung adenocarcinoma, low KLF10 expression associated with decreased patient survival, consistent with a pivotal role for KLF10 in distinguishing the antiproliferative versus prometastatic functions of TGFβ. Our results establish that KLF10 functions to suppress TGFβ-induced EMT, establishing a molecular basis for the dichotomy of TGFβ function during tumor progression. Cancer Res; 77(9); 2387–400. ©2017 AACR.</jats:p

Endoxifen’s Molecular Mechanisms of Action Are Concentration Dependent and Different than That of Other Anti-Estrogens

<div><p>Endoxifen, a cytochrome P450 mediated tamoxifen metabolite, is being developed as a drug for the treatment of estrogen receptor (ER) positive breast cancer. Endoxifen is known to be a potent anti-estrogen and its mechanisms of action are still being elucidated. Here, we demonstrate that endoxifen-mediated recruitment of ERα to known target genes differs from that of 4-hydroxy-tamoxifen (4HT) and ICI-182,780 (ICI). Global gene expression profiling of MCF7 cells revealed substantial differences in the transcriptome following treatment with 4HT, endoxifen and ICI, both in the presence and absence of estrogen. Alterations in endoxifen concentrations also dramatically altered the gene expression profiles of MCF7 cells, even in the presence of clinically relevant concentrations of tamoxifen and its metabolites, 4HT and N-desmethyl-tamoxifen (NDT). Pathway analysis of differentially regulated genes revealed substantial differences related to endoxifen concentrations including significant induction of cell cycle arrest and markers of apoptosis following treatment with high, but not low, concentrations of endoxifen. Taken together, these data demonstrate that endoxifen’s mechanism of action is different from that of 4HT and ICI and provide mechanistic insight into the potential importance of endoxifen in the suppression of breast cancer growth and progression.</p> </div

ChIP analysis of ERα binding to a consensus ERE and endogenous target genes.

<p>ChIP assays were performed in MCF7 cells transiently transfected with a consensus ERE and treated as indicated for either 1 hour (<b>A</b>) or 24 hours (<b>B</b>). Data are expressed as the relative abundance of the target following indicated treatments relative to vehicle treated controls as detected by real-time PCR. All data were normalized using input values. Experiments were conducted in triplicate and a representative data set is shown. Asterisks denote significance at the P<0.05 level (ANOVA) compared to vehicle controls. # denotes significant differences (P<0.05) between estrogen and anti-estrogen treatments.</p

Real-time PCR confirmation of selected genes whose expression levels were either increased (red) or decreased (green) by a specific anti-estrogen.

<p>Genes whose expression levels were determined to be specifically increased (red) or decreased (green) by only one of the three anti-estrogen treatments were randomly selected for confirmation of the microarray data. Darkly shaded bars depict relative fold changes from vehicle treated cells (dashed line) as detected by microarray analysis while lightly shaded bars depict fold change as detected by RT-PCR analysis. Solid lines represent the 1.5 fold cut-off used in the microarray analysis. Data represent the mean ± the standard error of three independent treatments. Asterisks denote values with significant differences at the P<0.05 level (ANOVA) relative to vehicle treated controls which also met the 1.5 fold cut-off parameter used in the microarray analysis.</p

Venn diagrams depicting the anti-estrogen specific effects on estrogen-dependent and -independent genes.

<p>(<b>A</b>) Venn of genes whose expression levels were significantly altered by 1.5 fold or greater in MCF7 cells treated with 10 nM estrogen plus 100 nM concentrations of indicated anti-estrogens, relative to cells treated with estrogen alone following 24 hours of exposure. (<b>B</b>) Venn diagram of genes whose expression levels were significantly altered by 1.5 fold or greater in MCF7 cells treated with 10 nM estrogen plus 100 nM concentrations of indicated anti-estrogens, but not by estrogen treatment alone, relative to vehicle treated controls following 24 hours of exposure.</p

Pathway analysis of genes regulated by 100 nM or 20 nM endoxifen treatments in the presence of estrogen and physiologically relevant levels tamoxifen and its metabolites.

<p> The sub-categories of biological pathways determined to be significantly altered and which were unique to the 100 nM endoxifen treatment (<b>A</b>) or the 20 nM endoxifen treatment (<b>B</b>), or which were commonly regulated by both endoxifen concentrations (<b>C</b>), are shown. The specific biological pathways which are comprised within these sub-categories are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054613#pone-0054613-t001" target="_blank">Table 1</a>.</p

Profile of cell cycle changes induced by endoxifen.

<p>MCF7 cells were treated as indicated for 24 hours and cell cycle profiles were determined by propidium iodide staining and flow cytometry. (<b>A</b>) The percentage of cells from each treatment in G2/M phase (blue), S phase (green) and G1 phase (red) are shown. Asterisks within each cell cycle phase denote significance at the P<0.05 level (ANOVA) compared to vehicle controls. # within each cell cycle phase denotes significant differences (P<0.05) compared to estrogen treated cells. (<b>B</b>) Representative flow cytometry plots for each treatment condition.</p

Biological pathways detected to be significantly regulated based upon endoxifen concentrations under physiologically relevant conditions.

<p>Biological pathways detected to be significantly regulated based upon endoxifen concentrations under physiologically relevant conditions.</p