Zeb2 Regulates Cell Fate at the Exit from Epiblast State in Mouse Embryonic Stem Cells.

In human embryonic stem cells (ESCs) the transcription factor Zeb2 regulates neuroectoderm versus mesendoderm formation, but it is unclear how Zeb2 affects the global transcriptional regulatory network in these cell-fate decisions. We generated Zeb2 knockout (KO) mouse ESCs, subjected them as embryoid bodies (EBs) to neural and general differentiation and carried out temporal RNA-sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS) analysis in neural differentiation. This shows that Zeb2 acts preferentially as a transcriptional repressor associated with developmental progression and that Zeb2 KO ESCs can exit from their naïve state. However, most cells in these EBs stall in an early epiblast-like state and are impaired in both neural and mesendodermal differentiation. Genes involved in pluripotency, epithelial-to-mesenchymal transition (EMT), and DNA-(de)methylation, including Tet1, are deregulated in the absence of Zeb2. The observed elevated Tet1 levels in the mutant cells and the knowledge of previously mapped Tet1-binding sites correlate with loss-of-methylation in neural-stimulating conditions, however, after the cells initially acquired the correct DNA-methyl marks. Interestingly, cells from such Zeb2 KO EBs maintain the ability to re-adapt to 2i + LIF conditions even after prolonged differentiation, while knockdown of Tet1 partially rescues their impaired differentiation. Hence, in addition to its role in EMT, Zeb2 is critical in ESCs for exit from the epiblast state, and links the pluripotency network and DNA-methylation with irreversible commitment to differentiation. Stem Cells 2017;35:611-625.

Thyroid follicle development requires Smad1/5- and endothelial cell-dependent basement membrane assembly.

Thyroid follicles, the functional units of the thyroid gland, are delineated by a monolayer of thyrocytes resting on a continuous basement membrane. The developmental mechanisms of folliculogenesis, whereby follicles are formed by the reorganization of a non-structured mass of non-polarized epithelial cells, are largely unknown. Here we show that assembly of the epithelial basement membrane is crucial for folliculogenesis and is controlled by endothelial cell invasion and by BMP-Smad signaling in thyrocytes. Thyroid-specific Smad1 and Smad5 double-knockout (Smad1/5(dKO)) mice displayed growth retardation, hypothyroidism and defective follicular architecture. In Smad1/5(dKO) embryonic thyroids, epithelial cells remained associated in large clusters and formed small follicles. Although similar follicular defects are found in Vegfa knockout (Vegfa(KO)) thyroids, Smad1/5(dKO) thyroids had normal endothelial cell density yet impaired endothelial differentiation. Interestingly, both Vegfa(KO) and Smad1/5(dKO) thyroids displayed impaired basement membrane assembly. Furthermore, conditioned medium (CM) from embryonic endothelial progenitor cells (eEPCs) rescued the folliculogenesis defects of both Smad1/5(dKO) and Vegfa(KO) thyroids. Laminin α1, ß1 and γ1, abundantly released by eEPCs into CM, were crucial for folliculogenesis. Thus, epithelial Smad signaling and endothelial cell invasion promote folliculogenesis via assembly of the basement membrane.

BMP-SMAD Signaling Regulates Lineage Priming, but Is Dispensable for Self-Renewal in Mouse Embryonic Stem Cells.

Naive mouse embryonic stem cells (mESCs) are in a metastable state and fluctuate between inner cell mass- and epiblast-like phenotypes. Here, we show transient activation of the BMP-SMAD signaling pathway in mESCs containing a BMP-SMAD responsive reporter transgene. Activation of the BMP-SMAD reporter transgene in naive mESCs correlated with lower levels of genomic DNA methylation, high expression of 5-methylcytosine hydroxylases Tet1/2 and low levels of DNA methyltransferases Dnmt3a/b. Moreover, naive mESCs, in which the BMP-SMAD reporter transgene was activated, showed higher resistance to differentiation. Using double Smad1;Smad5 knockout mESCs, we showed that BMP-SMAD signaling is dispensable for self-renewal in both naive and ground state. These mutant mESCs were still pluripotent, but they exhibited higher levels of DNA methylation than their wild-type counterparts and had a higher propensity to differentiate. We showed that BMP-SMAD signaling modulates lineage priming in mESCs, by transiently regulating the enzymatic machinery responsible for DNA methylation.

Smad1/5 and Smad4 expression are important for osteoclast differentiation.

To investigate the necessity of the canonical BMP pathway during osteoclast differentiation, we created osteoclasts with a conditional gene deletion for Smad1 and Smad5 (SMAD1/5), or Smad4 using adenovirus expressing CRE recombinase (Ad-CRE). Reduction of either Smad4 or Smad1/5 expression resulted in fewer and smaller multinuclear cells compared to control cells. We also detected changes in osteoclast enriched genes, demonstrated by decreased Dc-stamp and cathepsin K expression in both Smad4 and Smad1/5 Ad-CRE osteoclasts, and changes in c-fos and Nfatc1 expression in only Smad4 Ad-CRE cells. Lastly we also detected a significant decrease in resorption pits and area resorbed in both the Smad4 and Smad1/5 Ad-CRE osteoclasts. Because we inhibited osteoclast differentiation with loss of either Smad4 or Smad1/5 expression, we assessed whether BMPs affected osteoclast activity in addition to BMP's effects on differentiation. Therefore, we treated mature osteoclasts with BMP2 or with dorsomorphin, a chemical inhibitor that selectively suppresses canonical BMP signaling. We demonstrated that BMP2 stimulated resorption in mature osteoclasts whereas treatment with dorsomorphin blocks osteoclast resorption. These results indicate that the BMP canonical signaling pathway is important for osteoclast differentiation and activity.

TDP2-dependent non-homologous end-joining protects against topoisomerase II-induced DNA breaks and genome instability in cells and in vivo.

Anticancer topoisomerase "poisons" exploit the break-and-rejoining mechanism of topoisomerase II (TOP2) to generate TOP2-linked DNA double-strand breaks (DSBs). This characteristic underlies the clinical efficacy of TOP2 poisons, but is also implicated in chromosomal translocations and genome instability associated with secondary, treatment-related, haematological malignancy. Despite this relevance for cancer therapy, the mechanistic aspects governing repair of TOP2-induced DSBs and the physiological consequences that absent or aberrant repair can have are still poorly understood. To address these deficits, we employed cells and mice lacking tyrosyl DNA phosphodiesterase 2 (TDP2), an enzyme that hydrolyses 5'-phosphotyrosyl bonds at TOP2-associated DSBs, and studied their response to TOP2 poisons. Our results demonstrate that TDP2 functions in non-homologous end-joining (NHEJ) and liberates DSB termini that are competent for ligation. Moreover, we show that the absence of TDP2 in cells impairs not only the capacity to repair TOP2-induced DSBs but also the accuracy of the process, thus compromising genome integrity. Most importantly, we find this TDP2-dependent NHEJ mechanism to be physiologically relevant, as Tdp2-deleted mice are sensitive to TOP2-induced damage, displaying marked lymphoid toxicity, severe intestinal damage, and increased genome instability in the bone marrow. Collectively, our data reveal TDP2-mediated error-free NHEJ as an efficient and accurate mechanism to repair TOP2-induced DSBs. Given the widespread use of TOP2 poisons in cancer chemotherapy, this raises the possibility of TDP2 being an important etiological factor in the response of tumours to this type of agent and in the development of treatment-related malignancy.

Robustness in angiogenesis: notch and BMP shaping waves.

Vascular patterning involves sprouting of blood vessels, which is governed by orchestrated communication between cells in the surrounding tissue and endothelial cells (ECs) lining the blood vessels. Single ECs are selected for sprouting by hypoxia-induced stimuli and become the 'tip' or leader cell that guides new sprouts. The 'stalk' or trailing ECs proliferate for tube extension and lumenize the nascent vessel. Stalk and tip cells can dynamically switch their identities during this process in a Notch-dependent manner. Here, we review recent studies showing that bone morphogenetic protein (BMP) signaling coregulates Notch target genes in ECs. In particular, we focus on how Delta-like ligand 4 (DLL4)-Notch and BMP effector interplay may drive nonsynchronized oscillatory gene expression in ECs essential for setting sharp tip-stalk cell boundaries while sustaining a dynamic pool of nonsprouting ECs. Deeper knowledge about the coregulation of vessel plasticity in different vascular beds may result in refinement of anti-angiogenesis and vessel normalization therapies.

TDP2 promotes repair of topoisomerase I-mediated DNA damage in the absence of TDP1.

The abortive activity of topoisomerases can result in clastogenic and/or lethal DNA damage in which the topoisomerase is covalently linked to the 3'- or 5'-terminus of a DNA strand break. This type of DNA damage is implicated in chromosome translocations and neurological disease and underlies the clinical efficacy of an important class of anticancer topoisomerase 'poisons'. Tyrosyl DNA phosphodiesterase-1 protects cells from abortive topoisomerase I (Top1) activity by hydrolyzing the 3'-phosphotyrosyl bond that links Top1 to a DNA strand break and is currently the only known human enzyme that displays this activity in cells. Recently, we identified a second tyrosyl DNA phosphodiesterase (TDP2; aka TTRAP/EAPII) that possesses weak 3'-tyrosyl DNA phosphodiesterase (3'-TDP) activity, in vitro. Herein, we have examined whether TDP2 contributes to the repair of Top1-mediated DNA breaks by deleting Tdp1 and Tdp2 separately and together in murine and avian cells. We show that while deletion of Tdp1 in wild-type DT40 cells and mouse embryonic fibroblasts decreases DNA strand break repair rates and cellular survival in response to Top1-induced DNA damage, deletion of Tdp2 does not. However, deletion of both Tdp1 and Tdp2 reduces rates of DNA strand break repair and cell survival below that observed in Tdp1-/- cells, suggesting that Tdp2 contributes to cellular 3'-TDP activity in the absence of Tdp1. Consistent with this idea, over-expression of human TDP2 in Tdp1-/-/Tdp2-/-/- DT40 cells increases DNA strand break repair rates and cell survival above that observed in Tdp1-/- DT40 cells, suggesting that Tdp2 over-expression can partially complement the defect imposed by loss of Tdp1. Finally, mice lacking both Tdp1 and Tdp2 exhibit greater sensitivity to Top1 poisons than do mice lacking Tdp1 alone, further suggesting that Tdp2 contributes to the repair of Top1-mediated DNA damage in the absence of Tdp1. In contrast, we failed to detect a contribution for Tdp1 to repair Top2-mediated damage. Together, our data suggest that Tdp1 and Tdp2 fulfil overlapping roles following Top1-induced DNA damage, but not following Top2-induced DNA damage, in vivo.

Stalk cell phenotype depends on integration of Notch and Smad1/5 signaling cascades.

Gradients of vascular endothelial growth factor (VEGF) induce single endothelial cells to become leading tip cells of emerging angiogenic sprouts. Tip cells then suppress tip-cell features in adjacent stalk cells via Dll4/Notch-mediated lateral inhibition. We report here that Smad1/Smad5-mediated BMP signaling synergizes with Notch signaling during selection of tip and stalk cells. Endothelium-specific inactivation of Smad1/Smad5 in mouse embryos results in impaired Dll4/Notch signaling and increased numbers of tip-cell-like cells at the expense of stalk cells. Smad1/5 downregulation in cultured endothelial cells reduced the expression of several target genes of Notch and of other stalk-cell-enriched transcripts (Hes1, Hey1, Jagged1, VEGFR1, and Id1-3). Moreover, Id proteins act as competence factors for stalk cells and form complexes with Hes1, which augment Hes1 levels in the endothelium. Our findings provide in vivo evidence for a regulatory loop between BMP/TGFß-Smad1/5 and Notch signaling that orchestrates tip- versus stalk-cell selection and vessel plasticity.

The EMT regulator Zeb2/Sip1 is essential for murine embryonic hematopoietic stem/progenitor cell differentiation and mobilization.

Zeb2 (Sip1/Zfhx1b) is a member of the zinc-finger E-box-binding (ZEB) family of transcriptional repressors previously demonstrated to regulate epithelial-to-mesenchymal transition (EMT) processes during embryogenesis and tumor progression. We found high Zeb2 mRNA expression levels in HSCs and hematopoietic progenitor cells (HPCs), and examined Zeb2 function in hematopoiesis through a conditional deletion approach using the Tie2-Cre and Vav-iCre recombination mouse lines. Detailed cellular analysis demonstrated that Zeb2 is dispensable for hematopoietic cluster and HSC formation in the aorta-gonadomesonephros region of the embryo, but is essential for normal HSC/HPC differentiation. In addition, Zeb2-deficient HSCs/HPCs fail to properly colonize the fetal liver and/or bone marrow and show enhanced adhesive properties associated with increased ß1 integrin and Cxcr4 expression. Moreover, deletion of Zeb2 resulted in embryonic (Tie2-Cre) and perinatal (Vav-icre) lethality due to severe cephalic hemorrhaging and decreased levels of angiopoietin-1 and, subsequently, improper pericyte coverage of the cephalic vasculature. These results reveal essential roles for Zeb2 in embryonic hematopoiesis and are suggestive of a role for Zeb2 in hematopoietic-related pathologies in the adult.

Smad1 and its target gene Wif1 coordinate BMP and Wnt signaling activities to regulate fetal lung development.

Bone morphogenetic protein 4 (Bmp4) is essential for lung development. To define the intracellular signaling mechanisms by which Bmp4 regulates lung development, BMP-specific Smad1 or Smad5 was selectively knocked out in fetal mouse lung epithelial cells. Abrogation of lung epithelial-specific Smad1, but not Smad5, resulted in retardation of lung branching morphogenesis and reduced sacculation, accompanied by altered distal lung epithelial cell proliferation and differentiation and, consequently, severe neonatal respiratory failure. By combining cDNA microarray with ChIP-chip analyses, Wnt inhibitory factor 1 (Wif1) was identified as a novel target gene of Smad1 in the developing mouse lung epithelial cells. Loss of Smad1 transcriptional activation of Wif1 was associated with reduced Wif1 expression and increased Wnt/ß-catenin signaling activity in lung epithelia, resulting in specific fetal lung abnormalities. This suggests a novel regulatory loop of Bmp4-Smad1-Wif1-Wnt/ß-catenin in coordinating BMP and Wnt pathways to control fetal lung development.

Loss of Smad5 leads to the disassembly of the apical junctional complex and increased susceptibility to experimental colitis.

The regulation of intestinal epithelial cell adhesion and migratory properties is often compromised in inflammatory bowel disease (IBD). Despite an increasing interest in bone morphogenetic protein (Bmp) signaling in gut pathologies, little is known of the specific roles played by individual Smads in intestinal epithelial functions. In the present study, we generated a mouse model with deletion of Smad5 transcriptional effector of the Bmp signaling pathway exclusively in the intestinal epithelium. Proliferation, migration, and apical junctional complex (AJC) protein expression were analyzed by immunofluorescence and Western blot. Human intestinal biopsies from control and IBD patients were analyzed for SMAD5 gene transcript expression by quantitative PCR (qPCR). Smad5(ΔIEC) and control mice were subjected to dextran sulfate sodium (DSS)-induced experimental colitis, and their clinical and histological symptoms were assessed. Loss of Smad5 led to intestinal epithelial hypermigration and deregulation of the expression of claudin-1 and claudin-2. E-cadherin was found to be equally expressed but displaced from the AJC to the cytoplasm in Smad5(ΔIEC) mice. Analysis of SMAD5 gene expression in human IBD patient samples revealed a significant downregulation of the gene transcript in Crohn's disease and ulcerative colitis samples. Smad5(ΔIEC) mice exposed to experimental DSS colitis were significantly more susceptible to the disease and had impaired wound healing during the recovery phase. Our results support that Smad5 is partly responsible for mediating Bmp signals in intestinal epithelial cells. In addition, deficiency in epithelial Smad5 leads to the deregulation of cell migration by disassembling the AJC with increasing susceptibility to experimental colitis and impairment in wound healing.

Essential validation of gene trap mouse ES cell lines: a test case with the gene Ttrap.

Gene trapping in mouse embryonic stem (ES) cells enables near-saturation vector-based insertional mutagenesis across the genome of this model organism. About 135,000 trapped ES cell lines are made available to the scientific community by the International Gene Trap Consortium (IGTC; www.genetrap.org). A search of one of its databases identified an ES cell line (RRS512) with a betaGeo-based gene trap (gt) vector insertion in intron 5 of Ttrap, a gene that encodes an intracellular signalling protein, which is implicated in gastrulation movement and left-right asymmetry in zebrafish embryos. We have determined the exact gt insertion point in the mutant ES cell clone RRS512 and confirmed the production of a chimaeric transcript consisting of the upstream Ttrap exons and the gene trap vector encoded marker/selection fusion sequences. This ES cell line was used to generate heterozygous Ttrap mutant mice, which were further crossed to obtain Ttrap(gt/gt) mice. In contrast to Ttraps documented essential role during nodal and Smad3 controlled zebrafish early embryogenesis, Ttrap(gt/gt) mice were born with a normal Mendelian distribution. However, subsequent analysis of these Ttrap(gt/gt) mice has revealed a duplication of the wild-type Ttrap allele that was already present in the RRS512 cell line. Based on our detailed analysis presented here, we suggest an extensive procedure for the characterization of gene trap ES cell lines prior to generating gene trap mice with these.

FGF-regulated BMP signaling is required for eyelid closure and to specify conjunctival epithelial cell fate.

There are conflicting reports about whether BMP signaling is required for eyelid closure during fetal development. This question was addressed using mice deficient in BMP or TGFbeta signaling in prospective eyelid and conjunctival epithelial cells. Genes encoding two type I BMP receptors, the type II TGFbeta receptor, two BMP- or two TGFbeta-activated R-Smads or the co-Smad Smad4 were deleted from the ocular surface ectoderm using Cre recombinase. Only mice with deletion of components of the BMP pathway had an 'eyelid open at birth' phenotype. Mice lacking Fgf10 or Fgfr2 also have open eyelids at birth. To better understand the pathways that regulate BMP expression and function during eyelid development, we localized BMPs and BMP signaling intermediates in Fgfr2 and Smad4 conditional knockout (CKO) mice. We found that Fgfr2 was required for the expression of Bmp4, the normal distribution of Shh signaling and for preserving the differentiation of the conjunctival epithelium. FGF signaling also promoted the expression of the Wnt antagonist Sfrp1 and suppressed Wnt signaling in the prospective eyelid epithelial cells, independently of BMP function. Transcripts encoding Foxc1 and Foxc2, which were previously shown to be necessary for eyelid closure, were not detectable in Smad4(CKO) animals. c-Jun, another key regulator of eyelid closure, was present and phosphorylated in eyelid periderm cells at the time of fusion, but failed to translocate to the nucleus in the absence of BMP function. Smad4(CKO) mice also showed premature differentiation of the conjunctival epithelium, conjunctival hyperplasia and the acquisition of epidermal characteristics, including formation of an ectopic row of hair follicles in place of the Meibomian glands. A second row of eyelashes is a feature of human lymphedema-distichiasis syndrome, which is associated with mutations in FOXC2.

Conditional deletion of Smad1 and Smad5 in somatic cells of male and female gonads leads to metastatic tumor development in mice.

The transforming growth factor beta (TGFbeta) family has critical roles in the regulation of fertility. In addition, the pathogenesis of some human cancers is attributed to misregulation of TGFbeta function and SMAD2 or SMAD4 mutations. There are limited mouse models for the BMP signaling SMADs (BR-SMADs) 1, 5, and 8 because of embryonic lethality and suspected genetic redundancy. Using tissue-specific ablation in mice, we deleted the BR-SMADs from somatic cells of ovaries and testes. Single conditional knockouts for Smad1 or Smad5 or mice homozygous null for Smad8 are viable and fertile. Female double Smad1 Smad5 and triple Smad1 Smad5 Smad8 conditional knockout mice become infertile and develop metastatic granulosa cell tumors. Male double Smad1 Smad5 conditional knockout mice are fertile but demonstrate metastatic testicular tumor development. Microarray analysis indicated significant alterations in expression of genes related to the TGFbeta pathway, as well as genes involved in infertility and extracellular matrix production. These data strongly implicate the BR-SMADs as part of a critical developmental pathway in ovaries and testis that, when disrupted, leads to malignant transformation.

Smad5 is dispensable for adult murine hematopoiesis.

Smad5 is known to transduce intracellular signals from bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-beta (TGF-beta) superfamily and are involved in the regulation of hematopoiesis. Recent findings suggest that BMP4 stimulates proliferation of human primitive hematopoietic progenitors in vitro, while early progenitors from mice deficient in Smad5 display increased self-renewal capacity in murine embryonic hematopoiesis. Here, we evaluate the role of Smad5 in the regulation of hematopoietic stem cell (HSC) fate decisions in adult mice by using an inducible MxCre-mediated conditional knockout model. Surprisingly, analysis of induced animals revealed unperturbed cell numbers and lineage distribution in peripheral blood (PB), bone marrow (BM), and the spleen. Furthermore, phenotypic characterization of the stem cell compartment revealed normal numbers of primitive lin(-)Sca-1(+)c-Kit(+) (LSK) cells in Smad5(-)(/)(-) BM. When transplanted in a competitive fashion into lethally irradiated primary and secondary recipients, Smad5-deficient BM cells competed normally with wild-type (wt) cells, were able to provide long-term reconstitution for the hosts, and displayed normal lineage distribution. Taken together, Smad5-deficient HSCs from adult mice show unaltered differentiation, proliferation, and repopulating capacity. Therefore, in contrast to its role in embryonic hematopoiesis, Smad5 is dispensable for hematopoiesis in the adult mouse.

Mice lacking Dfna5 show a diverging number of cochlear fourth row outer hair cells.

A complex mutation in DFNA5, resulting in exon 8 skipping, causes autosomal dominant hearing impairment, which starts in the high frequencies between 5 and 15 years of age and progressively affects all frequencies. To study its function in vivo, Dfna5 knockout mice were generated through the deletion of exon 8, simultaneously mimicking the human mutation. To test the hearing impairment, frequency-specific Auditory Brainstem Response (ABR) measurements were performed at different ages in two genetic backgrounds (C57Bl/6J and CBA/Ca), but no differences between Dfna5-/- and Dfna5+/+ mice could be demonstrated. Morphological studies demonstrated significant differences in the number of fourth row outer hair cells between Dfna5-/- mice and their wild-type littermates. These results were obtained in both genetic backgrounds, albeit with opposite effects. In contrast to the results obtained in Dfna5-/- zebrafish, we did not observe different UDP-glucose dehydrogenase and hyaluronic acid levels in Dfna5-/- mice when compared to Dfna5+/+ mice.