<i>Foxp1</i> deficiency augments the proportion of S-phase HFSC at anagen phase.

<p><b>A:</b> IHC with anti-CD34 (red) and anti-BrdU (green) staining of hair follicles following 4-day BrdU pulse-chase in the <i>Foxp1</i>^<i>fl/fl</i> (WT) and <i>K14-Cre; Foxp1</i>^<i>fl/fl</i> (<i>cKO</i>) mice (P20-P23). The upper panel showed the timing of BrdU injection and sectioning. Abbreviations: Bu, bulge; HG, hair germ; DP, dermal papillae. Scale bars: 25 μm. <b>B:</b> Quantification of the number of BrdU⁺ cells in the bulges of (A). The <i>cKO</i> hair follicles at P24 displayed extensive BrdU⁺ cells in the hair germ and bulge cells, whereas the WT controls had few BrdU⁺ cells in the identical regions (n = 3,4). *, p<0.05. <b>C:</b> IHC for hair follicles at P55 following 28-day BrdU pulse-chase. The upper panel showed the timing of BrdU injection and sectioning. Scale bars: 75 μm. <b>D:</b> Quantification of the percentages of LRC in the bulges of (C). Few label-retaining cells (LRC) were detected in the bulges of <i>Foxp1 cKO</i> mice. n = 4; *, p<0.05. <b>E</b>: NAC treatment and BrdU injection once a day from P23 to P26 enhanced cell proliferation of HFSCs in WT early anagen. Scale bar, 50μm. <b>F:</b> Quantification of the frequency of BrdU⁺ cells in HFSCs in (E). *, p<0.05. <b>G-H:</b> Western blotting demonstrated a decrease of Foxp1 (G) and p19^ARF (H) protein levels within <i>cKO</i> hair follicles at anagen (P23). <b>I:</b> Down-regulation of <i>p19</i>^<i>ARF</i> transcripts within <i>cKO</i> anagen (P23) hair follicles relative to the WT as determined by qRT-PCR. <b>J:</b> S15 phosphorylated-p53 protein level was relatively decreased within <i>cKO</i> anagen (P23) hair follicles.</p

The dynamic nucleocytoplasmic localization of Foxp1 in response to oxidative stress.

<p><b>A:</b> Identification by mass spectrum analysis of phosphorylation at Foxp1 S468 sensitive to 2-hour H₂O₂ stimulation in CHO cells. <b>B:</b> Alignment of Foxp1 N-terminal residues across multiple species revealed a conserved nuclear localization motif of RXXRS (boxed). <b>C:</b> Linear schematics of Foxp1 depicting mutations (in blue) of the RXXRS motif and S468. Zinc-finger (ZF), leucine-zipper (LZ) and forkhead domains (FHD) are indicated by boxes. In Foxp1N, RDTR is mutated to HDTG, leading to loss of function of NLS; in Foxp1(S468A), A is substituted for S, leading to loss of phosphorylation at S468. <b>D:</b> Representative images showing defective nuclear localization of the Foxp1NLSm-EGFP fusion protein (green) following transient transfection into HaCat cells. <b>E:</b> Representative images showing defective nuclear export of the Foxp1(S468A)-EGFP fusion protein in transfected HaCat cells following one-hour stimulation with 500 μM H₂O₂. Green, EGFP fluorescence; DAPI, blue; scale bar: 10 μm. <b>F:</b> 293T cells were transfected with Foxp1 or Foxp1(S468A) expression constructs. Western blot was conducted to evaluate the relative level of Foxp1 protein in cytoplasm or nucleus following one-hour stimulation with 500 μM H₂O₂. <b>G:</b> Quantification of the relative Foxp1 levels by gray scale in (F, n = 3).</p

Foxp1 tunes ROS level through protein interaction with Trx-1.

<p><b>A:</b> Anti-Trx1 IHC confirming extensive expression of Trx1 in HFs at anagen (P23), catagen (P40) and telogen (P55). <b>B:</b> Interaction of Foxp1 and Trx-1 endogenous proteins in anagen HFs as determined by Co-IP of protein lysates. <b>C:</b> Interaction of Foxp1 and Trx-1 following ectopic expression of Foxp1-His and Trx1 in transfected HeLa cells. Cell lysates were immunoprecipitated by anti-Trx1 antibody and detected by anti-His antibody. <b>D:</b> Colocalization of Trx1-RFP and Foxp1-EGFP protein within the nuclei (blue, DAPI) of HaCat cells. <b>E:</b> Flow cytometry of DCFDA-stained HEK293T cells following transient transfection of the indicated constructs (2 μg Foxp1 and/or 2 μg Trx1 expressing vector) indicated that Foxp1 releases inhibition of Trx-1-mediated ROS accrual. <b>F:</b> Model for the mechanism by which Foxp1 regulates redox homeostasis during hair cycling. Foxp1 is located within nuclei under conditions of low oxidative stress. Foxp1 suppresses the function of the Trx1 protein in decreasing ROS levels, and then imposes cell cycle arrest through p19/p53 axis. Foxp1 is exported into the cytoplasm when the ROS levels approach a high threshold.</p

Foxp1 is translocated from the nucleus to cytoplasm of HFSCs at phases from anagen to catagen accompanying with rise of ROS levels.

<p><b>A:</b> Foxp1 distribution among distinct populations of hair follicles. Foxp1 was localized within nuclei from anagen I to III, exported to cytoplasm from catagen I to catagen V, and progressively relocalized into the nuclei at catagen VI and telogen. Lower panel was the high power view of upper panel. Scale bars: 50 μm. Blue, DAPI; red, anti-Foxp1. Abbreviations: Ep, epidermis; IFE, interfollicular epidermis; Bu, bulge; HG, hair germ; DP, dermal papillae; ORS, outer root sheath; IRS, inner root sheath; HF: hair shaft. <b>B:</b> Representative dot plot of FACS for HFSCs identified by CD34⁺/Integrin α6⁺ at early telogen (P49). Abbreviations: HFSCs, hair follicle stem cells. <b>C:</b> Histograms of DCFDA fluorescence intensities of HFSCs at telogen (P20), early anagen (P24), late anagen (P27) and catagen (P40) (n = 6, 6, 7, and 6, respectively). <b>D:</b> Quantification of (B) indicates a progressive increase in ROS levels in HFSCs from telogen to catagen. *, p<0.05; **, p<0.01.</p

Smyd1 Facilitates Heart Development by Antagonizing Oxidative and ER Stress Responses

<div><p>Smyd1/Bop is an evolutionary conserved histone methyltransferase previously shown by conventional knockout to be critical for embryonic heart development. To further explore the mechanism(s) in a cell autonomous context, we conditionally ablated <i>Smyd1</i> in the first and second heart fields of mice using a knock-in (KI) <i>Nkx2</i>.<i>5-cre</i> driver. Robust deletion of <i>floxed-Smyd1</i> in cardiomyocytes and the outflow tract (OFT) resulted in embryonic lethality at E9.5, truncation of the OFT and right ventricle, and additional defects consistent with impaired expansion and proliferation of the second heart field (SHF). Using a transgenic (Tg) <i>Nkx2</i>.<i>5-cre</i> driver previously shown to not delete in the SHF and OFT, early embryonic lethality was bypassed and both ventricular chambers were formed; however, reduced cardiomyocyte proliferation and other heart defects resulted in later embryonic death at E11.5-12.5. Proliferative impairment prior to both early and mid-gestational lethality was accompanied by dysregulation of transcripts critical for endoplasmic reticulum (ER) stress. Mid-gestational death was also associated with impairment of oxidative stress defense—a phenotype highly similar to the previously characterized knockout of the Smyd1-interacting transcription factor, skNAC. We describe a potential feedback mechanism in which the stress response factor Tribbles3/TRB3, when directly methylated by Smyd1, acts as a co-repressor of Smyd1-mediated transcription. Our findings suggest that Smyd1 is required for maintaining cardiomyocyte proliferation at minimally two different embryonic heart developmental stages, and its loss leads to linked stress responses that signal ensuing lethality.</p></div

Loss of <i>Smyd1</i> using <i>Ki-Nkx2</i>.<i>5</i>^<i>cre/+</i> disrupts looping morphogenesis and chamber formation through perturbation of the SHF and activation of ER stress.

<p><b>A.</b> Gross morphological comparison of control (Cx: <i>Nkx2</i>.<i>5</i>^<i>+/+</i>; <i>Smyd1</i>^{<i>Flox/Flox</i>}) and <i>Smyd1</i> Ki-CKO (CKO: <i>Nkx2</i>.<i>5</i>^<i>cre/+</i>; <i>Smyd1</i>^{<i>Flox/Flox</i>}) hearts at E9.5. Scale bar = 200 μm. <b>B, C</b>. The lengths of the outflow tract (OFT) (B) and right ventricle (RV) (C) were significantly reduced in Ki-CKO embryos at E9.5 (n = 6/group). <b>D.</b> Representative results of microarray gene expression comparison of transcripts critical to SHF and chamber formation at E9.5. Data are presented as expression values of 2 independent biological replicas of each genotype averaged from 2 technical replicas. <b>E.</b> Confirmation of microarray for deregulated transcripts critical to SHF and chamber formation by real-time PCR using RNA from E9.5 heart/pharyngeal mesoderm (n = 9/group). <b>F</b>. Whole mount <i>in situ</i> hybridization comparison of selected SHF and chamber formation transcripts deregulated and/or mislocalized in CKO hearts at E9.5 (n = 3/group). Arrows denote areas of differential expression. <b>G.</b> Comparison of cell proliferation in the outflow tract of Control (Cx) and Ki-CKO by BrdU immunohistochemistry. PE, Pharyngeal Endoderm. OFT, outflow tract. V, ventricle. Scale bar = 100 μm. <b>H.</b> Quantification of anti-BrdU staining in the outflow tract (n = 6/group). <b>I.</b> Loss of Smyd1 leads to deregulation of genes critical to anti-proliferative responses to ER stress. Data are presented as a heat map with expression values of 2 independent biological replicas of each genotype averaged from 2 technical replicas plotted as log² expression values. For B, C, E and H, data were analyzed by Student’s t-test (*P < 0.05).</p

Deletion of <i>Smyd1</i> by <i>Tg-Nkx2</i>.<i>5-cre</i> leads to a delayed embryonic lethal cardiac phenotype.

<p><b>A.</b><i>Smyd1</i>^{<i>flox/flox</i>}; <i>Tg-Nkx2</i>.<i>5-cre</i> (Tg-CKO) embryos die at midgestation. Table numbers are total recovered embryos of each genotype. The number of dead or abnormal embryos is given in parentheses. <b>B, C.</b><i>Smyd1</i> mRNA expression at E10.5 assayed by RT-PCR (B) and real-time PCR (C). <b>D.</b> H&E-stained transverse sections of E11.5 control (Cx) and Tg-CKO embryos showing pericardial edema, thinned pericardium and decreased trabeculation. <b>E.</b> Decreased proliferation was observed in the hearts of E10.5 <i>Smyd1</i> Tg-CKO embryos. Representative images of Cx and Tg-CKO hearts stained with H&E (upper panels) and the mitosis marker phospho-histone H3 serine 10 (p-H3) (lower panels). <b>F.</b> Quantification of p-H3 positive cells within the heart from three sections for three independent embryos (n = 3). <b>G.</b> Comparison of <i>skNAC</i> knockout and <i>Smyd1 Tg-CKO</i> heart gene expression by real-time PCR at E11.5. Data, focused primarily on oxidative response deregulation, are presented as mean for each genotype (<i>skNAC</i>^-/-, n = 5; <i>Smyd1</i> Tg-CKO, n = 4). Error bars indicate SEM. <b>H.</b> Genes encoding mediators of ER stress are deregulated by loss of Smyd1. Real-time PCR data represents average of 3 biological replicates each with 3 technical replicates; error bars indicate SEM. Data were analyzed by Student’s t-test (*P < 0.05, **P <0.01, ***P < 0.001, ****P < 0.0001).</p

Expression of <i>Smyd1</i> during early heart development.

<p><b>A</b>. Whole mount <i>in situ</i> hybridization for <i>Smyd1</i> in E8.5 (top panel) and E9.5 embryos (bottom panel) are shown from the left and right side. Red arrows denote the boundaries of <i>Smyd1</i> expression at the atrial and venous poles. Scale bar = 500 μm. <b>B.</b><i>In situ</i> hybridization for <i>Smyd1</i> in transverse sections of E10.0 hearts. The box in the left panel is enlarged in the right panel. <i>Smyd1</i> mRNA was enriched in the myocardium and not detectable in either the epicardium or endocardium. Black arrow = epicardium; Black arrowhead = endocardium. <b>C.</b> Immunolocalization of Smyd1 protein in the heart at E13.5. Smyd1 was specifically detected in cardiomyocytes. No Smyd1 protein was detectable in the endocardium (red arrowhead), epicardium (yellow arrowhead) or coronary vasculature (white arrowhead). Scale bar = 50 μm.</p

An Integrated Cell Purification and Genomics Strategy Reveals Multiple Regulators of Pancreas Development

<div><p>The regulatory logic underlying global transcriptional programs controlling development of visceral organs like the pancreas remains undiscovered. Here, we profiled gene expression in 12 purified populations of fetal and adult pancreatic epithelial cells representing crucial progenitor cell subsets, and their endocrine or exocrine progeny. Using probabilistic models to decode the general programs organizing gene expression, we identified co-expressed gene sets in cell subsets that revealed patterns and processes governing progenitor cell development, lineage specification, and endocrine cell maturation. Purification of <i>Neurog3</i> mutant cells and module network analysis linked established regulators such as <i>Neurog3</i> to unrecognized gene targets and roles in pancreas development. Iterative module network analysis nominated and prioritized transcriptional regulators, including diabetes risk genes. Functional validation of a subset of candidate regulators with corresponding mutant mice revealed that the transcription factors <i>Etv1</i>, <i>Prdm16</i>, <i>Runx1t1</i> and <i>Bcl11a</i> are essential for pancreas development. Our integrated approach provides a unique framework for identifying regulatory genes and functional gene sets underlying pancreas development and associated diseases such as diabetes mellitus.</p></div

Gene-module network reveals candidate pancreas regulators.

<p>(A) Normalized expression values of Prdm16 in sorted cells. (B) Normalized expression values of Bcl11a from purified cell populations. (C) Relative mRNA expression in Bcl11a mutant mice (n = 4) and control mice (n = 4) in sorted cells enriched for endocrine cells at E15. (D) Normalized expression values for Etv1 from purified cell populations. (E) Relative mRNA expression of pancreatic markers in Etv1 mutant (n = 4) and control (n = 4) pancreata at E18. (F) Cell mass changes in PP cells in Etv1 mutant mice at birth (n = 3). (G) Normalized expression values for Runx1t1 from purified cell populations. (H) Relative gene expression in Runx1t1 mutant mice (n = 4) and controls (n = 4) at E18 from whole pancreata. In (B–G), data are represented as mean +/− SEM. In (C), (E), (H) expression levels were normalized to <i>beta-actin</i> and results are shown relative to littermate controls, (A), (B), (D), (G) represent raw values obtained from microarray analysis.</p