Interplay between the EMT transcription factors ZEB1 and ZEB2 regulates hematopoietic stem and progenitor cell differentiation and hematopoietic lineage fidelity.

The ZEB2 transcription factor has been demonstrated to play important roles in hematopoiesis and leukemic transformation. ZEB1 is a close family member of ZEB2 but has remained more enigmatic concerning its roles in hematopoiesis. Here, we show using conditional loss-of-function approaches and bone marrow (BM) reconstitution experiments that ZEB1 plays a cell-autonomous role in hematopoietic lineage differentiation, particularly as a positive regulator of monocyte development in addition to its previously reported important role in T-cell differentiation. Analysis of existing single-cell (sc) RNA sequencing (RNA-seq) data of early hematopoiesis has revealed distinctive expression differences between Zeb1 and Zeb2 in hematopoietic stem and progenitor cell (HSPC) differentiation, with Zeb2 being more highly and broadly expressed than Zeb1 except at a key transition point (short-term HSC [ST-HSC]➔MPP1), whereby Zeb1 appears to be the dominantly expressed family member. Inducible genetic inactivation of both Zeb1 and Zeb2 using a tamoxifen-inducible Cre-mediated approach leads to acute BM failure at this transition point with increased long-term and short-term hematopoietic stem cell numbers and an accompanying decrease in all hematopoietic lineage differentiation. Bioinformatics analysis of RNA-seq data has revealed that ZEB2 acts predominantly as a transcriptional repressor involved in restraining mature hematopoietic lineage gene expression programs from being expressed too early in HSPCs. ZEB1 appears to fine-tune this repressive role during hematopoiesis to ensure hematopoietic lineage fidelity. Analysis of Rosa26 locus-based transgenic models has revealed that Zeb1 as well as Zeb2 cDNA-based overexpression within the hematopoietic system can drive extramedullary hematopoiesis/splenomegaly and enhance monocyte development. Finally, inactivation of Zeb2 alone or Zeb1/2 together was found to enhance survival in secondary MLL-AF9 acute myeloid leukemia (AML) models attesting to the oncogenic role of ZEB1/2 in AML.

Local delivery of hrBMP4 as an anticancer therapy in patients with recurrent glioblastoma: a first-in-human phase 1 dose escalation trial.

BACKGROUND: This Phase 1 study evaluates the intra- and peritumoral administration by convection enhanced delivery (CED) of human recombinant Bone Morphogenetic Protein 4 (hrBMP4) - an inhibitory regulator of cancer stem cells (CSCs) - in recurrent glioblastoma. METHODS: In a 3 + 3 dose escalation design, over four to six days, fifteen recurrent glioblastoma patients received, by CED, one of five doses of hrBMP4 ranging from 0·5 to 18 mg. Patients were followed by periodic physical, neurological, blood testing, magnetic resonance imaging (MRI) and quality of life evaluations. The primary objective of this first-in-human study was to determine the safety, dose-limiting toxicities (DLT) and maximum tolerated dose (MTD) of hrBMP4. Secondary objectives were to assess potential efficacy and systemic exposure to hrBMP4 upon intracerebral infusion. RESULTS: Intra- and peritumoral infusion of hrBMP4 was safe and well-tolerated. We observed no serious adverse events related to this drug. Neither MTD nor DLT were reached. Three patients had increased hrBMP4 serum levels at the end of infusion, which normalized within 4 weeks, without sign of toxicity. One patient showed partial response and two patients a complete (local) tumor response, which was maintained until the most recent follow-up, 57 and 30 months post-hrBMP4. Tumor growth was inhibited in areas permeated by hrBMP4. CONCLUSION: Local delivery of hrBMP4 in and around recurring glioblastoma is safe and well-tolerated. Three patients responded to the treatment. A complete response and long-term survival occurred in two of them. This warrants further clinical studies on this novel treatment targeting glioblastoma CSCs. TRIAL REGISTRATION: ClinicaTrials.gov identifier: NCT02869243.

Multifaceted actions of Zeb2 in postnatal neurogenesis from the ventricular-subventricular zone to the olfactory bulb.

The transcription factor Zeb2 controls fate specification and subsequent differentiation and maturation of multiple cell types in various embryonic tissues. It binds many protein partners, including activated Smad proteins and the NuRD co-repressor complex. How Zeb2 subdomains support cell differentiation in various contexts has remained elusive. Here, we studied the role of Zeb2 and its domains in neurogenesis and neural differentiation in the young postnatal ventricular-subventricular zone (V-SVZ), in which neural stem cells generate olfactory bulb-destined interneurons. Conditional Zeb2 knockouts and separate acute loss- and gain-of-function approaches indicated that Zeb2 is essential for controlling apoptosis and neuronal differentiation of V-SVZ progenitors before and after birth, and we identified Sox6 as a potential downstream target gene of Zeb2. Zeb2 genetic inactivation impaired the differentiation potential of the V-SVZ niche in a cell-autonomous fashion. We also provide evidence that its normal function in the V-SVZ also involves non-autonomous mechanisms. Additionally, we demonstrate distinct roles for Zeb2 protein-binding domains, suggesting that Zeb2 partners co-determine neuronal output from the mouse V-SVZ in both quantitative and qualitative ways in early postnatal life.

Targeted chromatin conformation analysis identifies novel distal neural enhancers of ZEB2 in pluripotent stem cell differentiation.

The transcription factor zinc finger E-box binding protein 2 (ZEB2) controls embryonic and adult cell fate decisions and cellular maturation in many stem/progenitor cell types. Defects in these processes in specific cell types underlie several aspects of Mowat-Wilson syndrome (MOWS), which is caused by ZEB2 haplo-insufficiency. Human ZEB2, like mouse Zeb2, is located on chromosome 2 downstream of a ±3.5 Mb-long gene-desert, lacking any protein-coding gene. Using temporal targeted chromatin capture (T2C), we show major chromatin structural changes based on mapping in-cis proximities between the ZEB2 promoter and this gene desert during neural differentiation of human-induced pluripotent stem cells, including at early neuroprogenitor cell (NPC)/rosette state, where ZEB2 mRNA levels increase significantly. Combining T2C with histone-3 acetylation mapping, we identified three novel candidate enhancers about 500 kb upstream of the ZEB2 transcription start site. Functional luciferase-based assays in heterologous cells and NPCs reveal co-operation between these three enhancers. This study is the first to document in-cis Regulatory Elements located in ZEB2's gene desert. The results further show the usability of T2C for future studies of ZEB2 REs in differentiation and maturation of multiple cell types and the molecular characterization of newly identified MOWS patients that lack mutations in ZEB2 protein-coding exons.

Zeb2 regulates the balance between retinal interneurons and Müller glia by inhibition of BMP-Smad signaling.

The interplay between signaling molecules and transcription factors during retinal development is key to controlling the correct number of retinal cell types. Zeb2 (Sip1) is a zinc-finger multidomain transcription factor that plays multiple roles in central and peripheral nervous system development. Haploinsufficiency of ZEB2 causes Mowat-Wilson syndrome, a congenital disease characterized by intellectual disability, epilepsy and Hirschsprung disease. In the developing retina, Zeb2 is required for generation of horizontal cells and the correct number of interneurons; however, its potential function in controlling gliogenic versus neurogenic decisions remains unresolved. Here we present cellular and molecular evidence of the inhibition of Müller glia cell fate by Zeb2 in late stages of retinogenesis. Unbiased transcriptomic profiling of control and Zeb2-deficient early-postnatal retina revealed that Zeb2 functions in inhibiting Id1/2/4 and Hes1 gene expression. These neural progenitor factors normally inhibit neural differentiation and promote Müller glia cell fate. Chromatin immunoprecipitation (ChIP) supported direct regulation of Id1 by Zeb2 in the postnatal retina. Reporter assays and ChIP analyses in differentiating neural progenitors provided further evidence that Zeb2 inhibits Id1 through inhibition of Smad-mediated activation of Id1 transcription. Together, the results suggest that Zeb2 promotes the timely differentiation of retinal interneurons at least in part by repressing BMP-Smad/Notch target genes that inhibit neurogenesis. These findings show that Zeb2 integrates extrinsic cues to regulate the balance between neuronal and glial cell types in the developing murine retina.

Amniotic ectoderm expansion in mouse occurs via distinct modes and requires SMAD5-mediated signalling.

Upon gastrulation, the mammalian conceptus transforms rapidly from a simple bilayer into a multilayered embryo enveloped by its extra-embryonic membranes. Impaired development of the amnion, the innermost membrane, causes major malformations. To clarify the origin of the mouse amnion, we used single-cell labelling and clonal analysis. We identified four clone types with distinct clonal growth patterns in amniotic ectoderm. Two main types have progenitors in extreme proximal-anterior epiblast. Early descendants initiate and expand amniotic ectoderm posteriorly, while descendants of cells remaining anteriorly later expand amniotic ectoderm from its anterior side. Amniogenesis is abnormal in embryos deficient in the bone morphogenetic protein (BMP) signalling effector SMAD5, with delayed closure of the proamniotic canal, and aberrant amnion and folding morphogenesis. Transcriptomics of individual Smad5 mutant amnions isolated before visible malformations and tetraploid chimera analysis revealed two amnion defect sets. We attribute them to impairment of progenitors of the two main cell populations in amniotic ectoderm and to compromised cuboidal-to-squamous transition of anterior amniotic ectoderm. In both cases, SMAD5 is crucial for expanding amniotic ectoderm rapidly into a stretchable squamous sheet to accommodate exocoelom expansion, axial growth and folding morphogenesis.

Integrative and perturbation-based analysis of the transcriptional dynamics of TGFß/BMP system components in transition from embryonic stem cells to neural progenitors.

Cooperative actions of extrinsic signals and cell-intrinsic transcription factors alter gene regulatory networks enabling cells to respond appropriately to environmental cues. Signaling by transforming growth factor type ß (TGFß) family ligands (eg, bone morphogenetic proteins [BMPs] and Activin/Nodal) exerts cell-type specific and context-dependent transcriptional changes, thereby steering cellular transitions throughout embryogenesis. Little is known about coordinated regulation and transcriptional interplay of the TGFß system. To understand intrafamily transcriptional regulation as part of this system's actions during development, we selected 95 of its components and investigated their mRNA-expression dynamics, gene-gene interactions, and single-cell expression heterogeneity in mouse embryonic stem cells transiting to neural progenitors. Interrogation at 24 hour intervals identified four types of temporal gene transcription profiles that capture all stages, that is, pluripotency, epiblast formation, and neural commitment. Then, between each stage we performed esiRNA-based perturbation of each individual component and documented the effect on steady-state mRNA levels of the remaining 94 components. This exposed an intricate system of multilevel regulation whereby the majority of gene-gene interactions display a marked cell-stage specific behavior. Furthermore, single-cell RNA-profiling at individual stages demonstrated the presence of detailed co-expression modules and subpopulations showing stable co-expression modules such as that of the core pluripotency genes at all stages. Our combinatorial experimental approach demonstrates how intrinsically complex transcriptional regulation within a given pathway is during cell fate/state transitions.

Zeb2 Regulates Myogenic Differentiation in Pluripotent Stem Cells.

Skeletal muscle differentiation is triggered by a unique family of myogenic basic helix-loop-helix transcription factors, including MyoD, MRF-4, Myf-5, and Myogenin. These transcription factors bind promoters and distant regulatory regions, including E-box elements, of genes whose expression is restricted to muscle cells. Other E-box binding zinc finger proteins target the same DNA response elements, however, their function in muscle development and regeneration is still unknown. Here, we show that the transcription factor zinc finger E-box-binding homeobox 2 (Zeb2, Sip-1, Zfhx1b) is present in skeletal muscle tissues. We investigate the role of Zeb2 in skeletal muscle differentiation using genetic tools and transgenic mouse embryonic stem cells, together with single-cell RNA-sequencing and in vivo muscle engraftment capability. We show that Zeb2 over-expression has a positive impact on skeletal muscle differentiation in pluripotent stem cells and adult myogenic progenitors. We therefore propose that Zeb2 is a novel myogenic regulator and a possible target for improving skeletal muscle regeneration. The non-neural roles of Zeb2 are poorly understood.

Sip1 regulates the generation of the inner nuclear layer retinal cell lineages in mammals.

The transcription factor Sip1 (Zeb2) plays multiple roles during CNS development from early acquisition of neural fate to cortical neurogenesis and gliogenesis. In humans, SIP1 (ZEB2) haploinsufficiency leads to Mowat-Wilson syndrome, a complex congenital anomaly including intellectual disability, epilepsy and Hirschsprung disease. Here we uncover the role of Sip1 in retinogenesis. Somatic deletion of Sip1 from mouse retinal progenitors primarily affects the generation of inner nuclear layer cell types, resulting in complete loss of horizontal cells and reduced numbers of amacrine and bipolar cells, while the number of Muller glia is increased. Molecular analysis places Sip1 downstream of the eye field transcription factor Pax6 and upstream of Ptf1a in the gene network required for generating the horizontal and amacrine lineages. Intriguingly, characterization of differentiation dynamics reveals that Sip1 has a role in promoting the timely differentiation of retinal interneurons, assuring generation of the proper number of the diverse neuronal and glial cell subtypes that constitute the functional retina in mammals.

The EMT transcription factor Zeb2 controls adult murine hematopoietic differentiation by regulating cytokine signaling.

Epithelial-to-mesenchymal-transition (EMT) is critical for normal embryogenesis and effective postnatal wound healing, but is also associated with cancer metastasis. SNAIL, ZEB, and TWIST families of transcription factors are key modulators of the EMT process, but their precise roles in adult hematopoietic development and homeostasis remain unclear. Here we report that genetic inactivation of Zeb2 results in increased frequency of stem and progenitor subpopulations within the bone marrow (BM) and spleen and that these changes accompany differentiation defects in multiple hematopoietic cell lineages. We found no evidence that Zeb2 is critical for hematopoietic stem cell self-renewal capacity. However, knocking out Zeb2 in the BM promoted a phenotype with several features that resemble human myeloproliferative disorders, such as BM fibrosis, splenomegaly, and extramedullary hematopoiesis. Global gene expression and intracellular signal transduction analysis revealed perturbations in specific cytokine and cytokine receptor-related signaling pathways following Zeb2 loss, especially the JAK-STAT and extracellular signal-regulated kinase pathways. Moreover, we detected some previously unknown mutations within the human Zeb2 gene (ZFX1B locus) from patients with myeloid disease. Collectively, our results demonstrate that Zeb2 controls adult hematopoietic differentiation and lineage fidelity through widespread modulation of dominant signaling pathways that may contribute to blood disorders.

p120 Catenin-Mediated Stabilization of E-Cadherin Is Essential for Primitive Endoderm Specification.

E-cadherin-mediated cell-cell adhesion is critical for naive pluripotency of cultured mouse embryonic stem cells (mESCs). E-cadherin-depleted mESC fail to downregulate their pluripotency program and are unable to initiate lineage commitment. To further explore the roles of cell adhesion molecules during mESC differentiation, we focused on p120 catenin (p120ctn). Although one key function of p120ctn is to stabilize and regulate cadherin-mediated cell-cell adhesion, it has many additional functions, including regulation of transcription and Rho GTPase activity. Here, we investigated the role of mouse p120ctn in early embryogenesis, mESC pluripotency and early fate determination. In contrast to the E-cadherin-null phenotype, p120ctn-null mESCs remained pluripotent, but their in vitro differentiation was incomplete. In particular, they failed to form cystic embryoid bodies and showed defects in primitive endoderm formation. To pinpoint the underlying mechanism, we undertook a structure-function approach. Rescue of p120ctn-null mESCs with different p120ctn wild-type and mutant expression constructs revealed that the long N-terminal domain of p120ctn and its regulatory domain for RhoA were dispensable, whereas its armadillo domain and interaction with E-cadherin were crucial for primitive endoderm formation. We conclude that p120ctn is not only an adaptor and regulator of E-cadherin, but is also indispensable for proper lineage commitment.

Differentiation of Mouse Enteric Nervous System Progenitor Cells Is Controlled by Endothelin 3 and Requires Regulation of Ednrb by SOX10 and ZEB2.

BACKGROUND & AIMS: Maintenance and differentiation of progenitor cells in the developing enteric nervous system are controlled by molecules such as the signaling protein endothelin 3 (EDN3), its receptor (the endothelin receptor type B [EDNRB]), and the transcription factors SRY-box 10 (SOX10) and zinc finger E-box binding homeobox 2 (ZEB2). We used enteric progenitor cell (EPC) cultures and mice to study the roles of these proteins in enteric neurogenesis and their cross regulation. METHODS: We performed studies in mice with a Zeb2 loss-of-function mutation (Zeb2Δ) and mice carrying a spontaneous recessive mutation that prevents conversion of EDN3 to its active form (Edn3ls). EPC cultures issued from embryos that expressed only wild-type Zeb2 (Zeb2+/+ EPCs) or were heterozygous for the mutation (Zeb2Δ/+ EPCs) were exposed to EDN3; we analyzed the effects on cell differentiation using immunocytochemistry. In parallel, Edn3ls mice were crossed with Zeb2Δ/+mice; intestinal tissues were collected from embryos for immunohistochemical analyses. We investigated regulation of the EDNRB gene in transactivation and chromatin immunoprecipitation assays; results were validated in functional rescue experiments using transgenes expression in EPCs from retroviral vectors. RESULTS: Zeb2Δ/+ EPCs had increased neuronal differentiation compared to Zeb2+/+ cells. When exposed to EDN3, Zeb2+/+ EPCs continued expression of ZEB2 but did not undergo any neuronal differentiation. Incubation of Zeb2Δ/+ EPCs with EDN3, on the other hand, resulted in only partial inhibition of neuronal differentiation. This indicated that 2 copies of Zeb2 are required for EDN3 to prevent neuronal differentiation. Mice with combined mutations in Zeb2 and Edn3 (double mutants) had more severe enteric anomalies and increased neuronal differentiation compared to mice with mutations in either gene alone. The transcription factors SOX10 and ZEB2 directly activated the EDNRB promoter. Overexpression of EDNRB in Zeb2Δ/+ EPCs restored inhibition of neuronal differentiation, similar to incubation of Zeb2+/+ EPCs with EDN3. CONCLUSIONS: In studies of cultured EPCs and mice, we found that control of differentiation of mouse enteric nervous system progenitor cells by EDN3 requires regulation of Ednrb expression by SOX10 and ZEB2.

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.

The Notch intracellular domain integrates signals from Wnt, Hedgehog, TGFß/BMP and hypoxia pathways.

Notch signaling is a highly conserved signal transduction pathway that regulates stem cell maintenance and differentiation in several organ systems. Upon activation, the Notch receptor is proteolytically processed, its intracellular domain (NICD) translocates into the nucleus and activates expression of target genes. Output, strength and duration of the signal are tightly regulated by post-translational modifications. Here we review the intracellular post-translational regulation of Notch that fine-tunes the outcome of the Notch response. We also describe how crosstalk with other conserved signaling pathways like the Wnt, Hedgehog, hypoxia and TGFß/BMP pathways can affect Notch signaling output. This regulation can happen by regulation of ligand, receptor or transcription factor expression, regulation of protein stability of intracellular key components, usage of the same cofactors or coregulation of the same key target genes. Since carcinogenesis is often dependent on at least two of these pathways, a better understanding of their molecular crosstalk is pivotal.

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.

NLS-tagging: an alternative strategy to tag nuclear proteins.

The characterization of transcription factor complexes and their binding sites in the genome by affinity purification has yielded tremendous new insights into how genes are regulated. The affinity purification requires either the use of antibodies raised against the factor of interest itself or by high-affinity binding of a C- or N-terminally added tag sequence to the factor. Unfortunately, fusing extra amino acids to the termini of a factor can interfere with its biological function or the tag may be inaccessible inside the protein. Here, we describe an effective solution to that problem by integrating the 'tag' close to the nuclear localization sequence domain of the factor. We demonstrate the effectiveness of this approach with the transcription factors Fli-1 and Irf2bp2, which cannot be tagged at their extremities without loss of function. This resulted in the identification of novel proteins partners and a new hypothesis on the contribution of Fli-1 to hematopoiesis.

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.

Deletion of MgcRacGAP in the male germ cells impairs spermatogenesis and causes male sterility in the mouse.

MgcRacGAP (RACGAP1) is a GTPase Activating Protein (GAP), highly produced in the mouse embryonic brain and in the human and mouse post-natal testis. MgcRacGAP negatively controls the activity of Rac and Cdc42, which are key molecular switches acting on the microtubule and actin cytoskeleton and controlling various cell processes such as proliferation, adhesion and motility. Previous studies demonstrated that MgcRacGAP plays a critical role in the cytokinesis of somatic cells; hence homozygous inactivation of the gene in the mouse and mutation in Caenorhabditis elegans led to embryonic lethality due to the inability of MgcRacGAP-null embryos to assemble the central spindle and to complete cytokinesis. In the testis, the germ cells do not complete cytokinesis and remain connected as a syncytium throughout the entire process of spermatogenesis. Interestingly, MgcRacGAP was shown to locate to the intercellular bridges, connecting these germ cells. In order to determine the function(s) of MgcRacGAP in the male germline, we generated a conditional knock-out mouse using Stra8 promoter driven Cre recombinase to induce the specific deletion of MgcRacGAP in the pre-meiotic germ cells. We found that the absence of MgcRacGAP induced a germline depletion and male sterility. Consistent with the role of MgcRacGAP in the establishment of the cytoplasm constriction during cytokinesis of the somatic cells, we observed that MgcRacGAP deletion in the germ cells prevented the formation of the intercellular bridges and induced a proliferation arrest. While we assume that inherited homozygous loss of function mutations in MgcRacGAP would be lethal in human, de novo mutations in the testis might account for some cases of non-obstructive oligo- and/or azoo-spermia syndromes, whose genetic causes are altogether still poorly defined.

Onecut transcription factors act upstream of Isl1 to regulate spinal motoneuron diversification.

During development, spinal motoneurons (MNs) diversify into a variety of subtypes that are specifically dedicated to the motor control of particular sets of skeletal muscles or visceral organs. MN diversification depends on the coordinated action of several transcriptional regulators including the LIM-HD factor Isl1, which is crucial for MN survival and fate determination. However, how these regulators cooperate to establish each MN subtype remains poorly understood. Here, using phenotypic analyses of single or compound mutant mouse embryos combined with gain-of-function experiments in chick embryonic spinal cord, we demonstrate that the transcriptional activators of the Onecut family critically regulate MN subtype diversification during spinal cord development. We provide evidence that Onecut factors directly stimulate Isl1 expression in specific MN subtypes and are therefore required to maintain Isl1 production at the time of MN diversification. In the absence of Onecut factors, we observed major alterations in MN fate decision characterized by the conversion of somatic to visceral MNs at the thoracic levels of the spinal cord and of medial to lateral MNs in the motor columns that innervate the limbs. Furthermore, we identify Sip1 (Zeb2) as a novel developmental regulator of visceral MN differentiation. Taken together, these data elucidate a comprehensive model wherein Onecut factors control multiple aspects of MN subtype diversification. They also shed light on the late roles of Isl1 in MN fate decision.