A global role for KLF1 in erythropoiesis revealed by ChIP-seq in primary erythroid cells

KLF1 regulates a diverse suite of genes to direct erythroid cell differentiation from bipotent progenitors. To determine the local cis-regulatory contexts and transcription factor networks in which KLF1 operates, we performed KLF1 ChIP-seq in the mouse. We found at least 945 sites in the genome of E14.5 fetal liver erythroid cells which are occupied by endogenous KLF1. Many of these recovered sites reside in erythroid gene promoters such as Hbb-bl, but the majority are distant to any known gene. Our data suggests KLF1 directly regulates most aspects of terminal erythroid differentiation including production of alpha- and beta-globin protein chains, heme biosynthesis, coordination of proliferation and anti-apoptotic pathways, and construction of the red cell membrane and cytoskeleton by functioning primarily as a transcriptional activator. Additionally, we suggest new mechanisms for KLF1 cooperation with other transcription factors, in particular the erythroid transcription factor GATA1, to maintain homeostasis in the erythroid compartment

Selective inhibition of cancer cell self-renewal through a Quisinostat-histone H1.0 axis

Continuous cancer growth is driven by subsets of self-renewing malignant cells. Targeting of uncontrolled self-renewal through inhibition of stem cell-related signaling pathways has proven challenging. Here, we show that cancer cells can be selectively deprived of self-renewal ability by interfering with their epigenetic state. Re-expression of histone H1.0, a tumor-suppressive factor that inhibits cancer cell self-renewal in many cancer types, can be broadly induced by the clinically well-tolerated compound Quisinostat. Through H1.0, Quisinostat inhibits cancer cell self-renewal and halts tumor maintenance without affecting normal stem cell function. Quisinostat also hinders expansion of cells surviving targeted therapy, independently of the cancer types and the resistance mechanism, and inhibits disease relapse in mouse models of lung cancer. Our results identify H1.0 as a major mediator of Quisinostat's antitumor effect and suggest that sequential administration of targeted therapy and Quisinostat may be a broadly applicable strategy to induce a prolonged response in patients

Rapid screening of gene function by systemic delivery of morpholino oligonucleotides to live mouse embryos

Traditional gene targeting methods in mice are complex and time consuming, especially when conditional deletion methods are required. Here, we describe a novel technique for assessing gene function by injection of modified antisense morpholino oligonucleotides (MOs) into the heart of mid-gestation mouse embryos. After allowing MOs to circulate through the embryonic vasculature, target tissues were explanted, cultured and analysed for expression of key markers. We established proof-of-principle by partially phenocopying known gene knockout phenotypes in the fetal gonads (Stra8, Sox9) and pancreas (Sox9). We also generated a novel double knockdown of Gli1 and Gli2, revealing defects in Leydig cell differentiation in the fetal testis. Finally, we gained insight into the roles of Adamts19 and Ctrb1, genes of unknown function in sex determination and gonadal development. These studies reveal the utility of this method as a means of first-pass analysis of gene function during organogenesis before committing to detailed genetic analysis

Primary cilia function regulates the length of the embryonic trunk axis and urogenital field in mice

The issues of whether and how some organs coordinate their size and shape with the blueprint of the embryo axis, while others appear to regulate their morphogenesis autonomously, remain poorly understood. Mutations in Ift144, encoding a component of the trafficking machinery of primary cilia assembly, result in a range of embryo patterning defects, affecting the limbs, skeleton and neural system. Here, we show that embryos of the mouse mutant Ift144(twt) develop gonads that are larger than wildtype. Investigation of the early patterning of the urogenital ridge revealed that the anterior posterior domain of the gonad/mesonephros was extended at 10.5 dpc, with no change in the length of the metanephros. In XY embryos, this extension resulted in an increase in testis cord number. Moreover, we observed a concomitant extension of the trunk axis in both sexes, with no change in the length of the tail domain or somite number. Our findings support a model in which: (1) primary cilia regulate embryonic trunk elongation; (2) the length of the trunk axis determines the size of the urogenital ridges; and (3) the gonad domain is partitioned into a number of testis cords that depends on the available space, rather than being divided a predetermined number of times to generate a specific number of cords. (C) 2014 Elsevier Inc. All rights reserved

Overview of method: MO delivery by heart injection.

<p>(A) Experimental pipeline from harvest of embryos through to injection, culture and downstream analyses. Visualisation of heart injection protocol can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114932#pone.0114932.s001" target="_blank">S1 Video</a> and images B–E. The cocktail of dye and MO in PBS is delivered via injection into the left ventricle of the beating heart at 11.5 dpc (B). Dye can be visualised going around the embryonic vasculature (indicated by white arrows) (C, D) and into the head vasculature (D) before the whole embryo is coloured (E). Schematic of ventricle injection (F) and the embryonic gonad which is highly vascularised (G). Delivery of India ink and F-MO (indicated by white arrows) shows the compounds reaching the mesonephric plexus at 5 min post-injection (H; <i>n</i> = 3); after 30 min F-MO positive cells were observed in the gonad proper (I; <i>n</i> = 3). s = seconds; min = minutes; g = gonad; m = mesonephros; F-MO = carboxyfluorescein-labelled standard control morpholino oligonucleotide. Scale bars: E = 1 mm, H = 0.5 mm.</p

Partial phenocopy of known gene knockouts in gonad and pancreas.

<p>(A, B) STRA8 knockdown: IF showed knockdown of STRA8 (A) in Stra8MO-treated XX gonads. Nuclear localisation of meiosis markers (γH2AX (A) and SCP3 (B); indicated by white arrows; see inserts) was absent but germ cells were present (POU5F1 (B); see inserts) in XX Stra8MO-treated gonads. (C–E) Knockdown of SOX9 in the gonad: Western blot for SOX9 (relative to α-TUBULIN or β-ACTIN) showed a downregulation of SOX9 (C) after Sox9MO treatment in XY gonads (<i>n</i> = 3). Downregulation of expression of SOX9 target gene <i>Amh</i> (D) expression was observed by qRT-PCR (<i>n</i> = 8, 15, 11, 4). IF for AMH and HSD3β (E) showed that AMH staining was weaker in XY Sox9MO samples compared to XY controls and that HSD3β-positive FLCs were present but staining was weaker in XY Sox9MO-treated gonads. (F–I) Knockdown of SOX9 in the pancreas: qRT-PCR of Sox9Mo treated pancreata showed <i>Ins1</i> (F) was downregulated but <i>Pax6</i> (G) was unchanged (<i>n</i> = 5, 5, 5, 5). Quantification of PAX6/INS-positive cells revealed that PAX6-positive (H) and INS-positive (I) cell number was unaltered by Sox9MO treatment (<i>n</i> = 3, 4, 2, 2). Scale bars = 100 μM; cMO = control morpholino; xMO = morpholino targeting gene x. For Western blots SOX9 levels were normalised to α-TUBULIN or β-ACTIN loading controls and Sox9MO-treated XY gonads measured relative to cMO treated XY gonads with expression for each blot set to 1. Rel. Ab./control = Relative Abundance of SOX9 to α-TUBULIN or β-ACTIN. For all qRT-PCR levels are shown relative to <i>Tbp</i>, error = S.E.M. For cell quantification error = S.E.M. with individual counts plotted. * = p = 0.05, ** = p = 0.001, *** = p = 0.0001, ns = not statistically significant.</p

Double knockdown of <i>Gli1/Gli2</i> in XY gonads.

<p>(A–D) Knockdown of GLI1/GLI2 in the gonad: qRT-PCR showed that treatment with Gli1/Gli2MO (<i>n</i> = 6, 5, 5, 8) resulted in no significant downregulation in steroidogenic regulator <i>Sf1/Nr5a1</i> (A) but a significant downregulation in expression of steroidogenic pathway enzymes <i>Hsd3β</i> (B), <i>Cyp11a1</i> (C) and <i>Star</i> (D). No change was observed in <i>Nr5a1</i> expression in Gli1MO or Gli2MO knockdown (E, I). Similarly, there were no changes in expression of steroidogenic pathway enzymes <i>Hsd3β</i> (F, J), <i>Cyp11a1</i> (G, K) and <i>Star</i> (H, L) in Gli1MO (E–H; <i>n</i> = 6, 6, 7, 5) or Gli2MO (I–L; <i>n</i> = 8, 7, 4, 3) single knockdowns. IF showed Sertoli cells (AMH (M) and SOX9 (N)) and germ cells (POU5F1 (M)) were present in XY Gli1/Gli2MO treated gonads and no FOXL2-positive cells were observed (N). Steroidogenic <i>Hsd3β</i>-positive (M) and <i>Nr5a1</i>-positive (N) cells were still present in Gli1/Gli2MO treated XY gonads. Quantification (<i>n</i> = 2) of steroidogenic cells revealed no change in the number of HSD3β-positive Leydig cells (O; green) or SF1-positive/SOX9-negative pre-Leydig cells (O; red). There was a decrease in the number of SOX9-positve Sertoli cells in the Gli1/2MO treated XY gonads (O; yellow). Scale bars = 100 μM; cMO = control morpholino; xMO = morpholino targeting gene x. For all qRT-PCR levels are shown relative to <i>Tbp</i>, error = S.E.M. For cell quantification error = S.E.M. with individual counts plotted. * = p = 0.05, ** = p = 0.001, ns = not statistically significant.</p