[Show abstract][Hide abstract] ABSTRACT: The mir-34/449 family consists of six homologous miRNAs at three genomic loci. Redundancy of miR-34/449 miRNAs and their dominant expression in multiciliated epithelia suggest a functional significance in ciliogenesis. Here we report that mice deficient for all miR-34/449 miRNAs exhibited postnatal mortality, infertility and strong respiratory dysfunction caused by defective mucociliary clearance. In both mouse and Xenopus, miR-34/449-deficient multiciliated cells (MCCs) exhibited a significant decrease in cilia length and number, due to defective basal body maturation and apical docking. The effect of miR-34/449 on ciliogenesis was mediated, at least in part, by post-transcriptional repression of Cp110, a centriolar protein suppressing cilia assembly. Consistent with this, cp110 knockdown in miR-34/449-deficient MCCs restored ciliogenesis by rescuing basal body maturation and docking. Altogether, our findings elucidate conserved cellular and molecular mechanisms through which miR-34/449 regulate motile ciliogenesis.
[Show abstract][Hide abstract] ABSTRACT: The development of primordial germ cells (PGCs) involves several waves of epigenetic reprogramming. A major step is following specification and involves the transition from the stably suppressive histone modification H3K9me2 to the more flexible, still repressive H3K27me3, while PGCs are arrested in G2 phase of their cycle. The significance and underlying molecular mechanism of this transition were so far unknown. Here, we generated mutant mice for the Mad2l2 (Mad2B, Rev7) gene product, and found that they are infertile in both males and females. We demonstrated that Mad2l2 is essential for PGC, but not somatic development. PGCs were specified normally in Mad2l2(-/-) embryos, but became eliminated by apoptosis during the subsequent phase of epigenetic reprogramming. A majority of knockout PGCs failed to arrest in the G2 phase, and did not switch from a H3K9me2 to a H3K27me3 configuration. By the analysis of transfected fibroblasts we found that the interaction of Mad2l2 with the histone methyltransferases G9a and GLP lead to a downregulation of H3K9me2. The inhibitory binding of Mad2l2 to Cyclin dependent kinase 1 (Cdk1) could arrest the cell cycle in the G2 phase, and also allowed another histone methyltransferase, Ezh2, to upregulate H3K27me3. Together, these results demonstrate the potential of Mad2l2 in the regulation of both cell cycle and the epigenetic status. The function of Mad2l2 is essential in PGCs, and thus of high relevance for fertility.
[Show abstract][Hide abstract] ABSTRACT: Pluripotency requires the expression of the three core transcriptions factors Oct4, Sox2 and Nanog, as well as further, complementary proteins. The geminin protein is part of this network, and was shown to play a role in the regulation of DNA replication, the control of the cell cycle, and the acquisition of neural fate. It is highly expressed in the early embryo, in particular the epiblast and the early neural ectoderm, and also in pluripotent embryonic stem cells. The genetic inactivation of geminin resulted in lethality after the first few cell divisions, and thus prohibited the outgrowth of pluripotent cells. We established embryonic stem cells allowing the deletion of the geminin gene by induction of of Cre-recombinase with tamoxifen. Here, we show that geminin deficiency quickly leads to a loss of pluripotency, and to differentiation into the mesendodermal direction with high Oct4/low Sox2 levels. Simultaneous loss of geminin and induction of the neural lineage resulted in immediate apoptosis. These results suggested that in early development geminin functions via the co-expressed Sox2 gene. We found that the stem cell enhancer SRR2 of Sox2 is occupied by the activating esBAF complex in the presence of geminin, but becomes epigenetically repressed in its absence by the Polycomb repressive complex PRC2. The importance of geminin for Sox2 expression also explains the absolute requirement for geminin during the induction of pluripotency by OSKM viruses. In summary, geminin is required for Sox2 expression, and thus for the maintenance of totipotency, pluripotency and the early neural lineage.
PLoS ONE 01/2013; 8(9):e73826. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Primordial germ cells (PGCs) are induced in the epiblast early in mammalian development. They develop their specific fate separate from somatic cells by the generation of a unique transcriptional profile and by epigenetic modifications of histones and DNA. PGCs are related to pluripotent cells in many respects, both on a molecular and a cell biological level. Mimicking their in vivo development, PGCs can be derived in culture from pluripotent cells. Vice versa, PGCs can be converted in vitro into pluripotent embryonic germ cells. Recent evidence indicates that the derivation of pluripotent embryonic stem cells from explanted inner cell mass cells may pass through a germ cell-like state, but that this intermediate is not obligatory. In this review, we discuss PGC development and its relevance to pluripotency in mammalian embryos. We outline possibilities and problems connected to the application of in vitro-derived germ cells in reproductive medicine.
Journal of Molecular Medicine 05/2012; 90(7):753-61. · 4.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hox transcription factors are key determinants of antero-posterior identity and have been implicated in assigning positionally appropriate neuron subtypes in the neural tube. These roles inherently necessitate stringent control mechanisms that confine Hox protein activities to discrete spatiotemporal domains. Here, we provide evidence that the timing and rostro-caudal extent of Hoxb8 activity in the neural tube is tightly regulated by miR-196, a microRNA species encoded within three Hox gene clusters. In vitro and in vivo sensor-tracer analysis and transcription assays revealed that miR-196 activity restricts the caudal extent of Hoxb8 expression to the thoracic-lumbar intersect via 3' UTR-dependent negative regulation. Spatio-temporally inappropriate Hoxb8 activity, through relief of miR-196-mediated repression or direct misexpression, affected normal progression of motor neuron genesis by affecting generic motor neuron differentiation programs. In addition to uncovering a role for microRNA-dependent restriction of caudal Hox activities, these data thus indicate novel aspects of Hox-dependent neural tube patterning by revealing a requirement of temporal regulation of a generic neuronal specification program.
[Show abstract][Hide abstract] ABSTRACT: The re-replication inhibitor Geminin binds to several transcription factors including homeodomain proteins, and to members of the polycomb and the SWI/SNF complexes.
Here we describe the TATA-binding protein-like factor-interacting protein (TIPT) isoform 2, as a strong binding partner of Geminin. TIPT2 is widely expressed in mouse embryonic and adult tissues, residing both in cyto- and nucleoplasma, and enriched in the nucleolus. Like Geminin, also TIPT2 interacts with several polycomb factors, with the general transcription factor TBP (TATA box binding protein), and with the related protein TBPL1 (TRF2). TIPT2 synergizes with geminin and TBP in the activation of TATA box-containing promoters, and with TBPL1 and geminin in the activation of the TATA-less NF1 promoter. Geminin and TIPT2 were detected in the chromatin near TBP/TBPL1 binding sites.
Together, our study introduces a novel transcriptional regulator and its function in cooperation with chromatin associated factors and the basal transcription machinery.
[Show abstract][Hide abstract] ABSTRACT: Large numbers and quantities of different, small RNA molecules are present in the cytoplasm of animal and plant cells. One subclass of these molecules is represented by the noncoding microRNAs. Since their discovery in the 1990s a multitude of basic information has accumulated, which has identified their function in post-transcriptional control, either via degradation or translational inhibition of target mRNAs. This function is in most of the cases a finetuning of gene expression, working in parallel with transcriptional regulatory processes. MicroRNA expression profiles are highly dynamic during embryonic development and in adulthood. Misexpression of microRNAs can perturb embryogenesis, organogenesis, tissue homeostasis and the cell cycle. Evidence from gain- and loss-of function studies indicates roles for microRNAs in pathophysiologic states including cardiac hypertrophy, muscle dystrophy, hepatitis infection, diabetes, Parkinson syndrome, hematological malignancies and other types of cancer. In this review, we focus on studies addressing the role of various microRNAs in heart, muscle, liver, pancreas, central nervous system, and hematopoiesis.
Current Molecular Medicine 01/2009; 8(8):698-710. · 4.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The secreted Frizzled receptor related proteins (Sfrp's) belong to a protein family that comprises antagonists and modifiers of Wnt and BMP signalling. Here we report the isolation and expression pattern of the Sfrp gene "Sizzled" in the chick. Sizzled genes, as well as the closely related crescent genes, exist in the genomes of fishes, frogs and chicks, but not of mammals. The chicken Sizzled gene (Szl) is initially expressed in the anterior endoderm of gastrulating and early head fold embryos. An additional, separate expression domain develops at the posterior end of the embryo from the head process stage onwards. Szl transcripts are then detected in precardial mesodermal cells, are transiently transcribed in the straight heart tube, and later prominently in the splanchnic mesoderm surrounding the arterial pole of the developing heart, the anterior heart field. These cells are subsequently recruited to form the cardiac outflow tract. cSzl expression is downregulated when the septation of the outflow tract by neural crest derived cells begins.
[Show abstract][Hide abstract] ABSTRACT: The geminin protein functions both as a DNA rereplication inhibitor through association with Cdt1 and as a repressor of Hox gene transcription through the polycomb pathway. Here, we report that the functions of avian geminin are coordinated with and regulated by cell cycle-dependent nuclear-cytoplasmic shuttling. In S phase, geminin enters nuclei and inhibits both loading of the minichromosome maintenance (MCM) complex onto chromatin and Hox gene transcription. At the end of mitosis, geminin is exported from nuclei by the exportin protein Crm1 and is unavailable in the nucleus during the next G(1) phase, thus ensuring proper chromatin loading of the MCM complex and Hox gene transcription. This mechanism for regulating the functions of geminin adds to distinct mechanisms, such as protein degradation and ubiquitination, applied in other vertebrates.
Molecular and Cellular Biology 08/2007; 27(13):4737-44. · 5.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Geminin is a multifunctional protein. After DNA replication is initiated during a cell cycle, geminin binds to Cdt1, one of the key DNA replication licensing factors. This highly regulated interaction sequestrates Cdt1, thus preventing DNA rereplication in the same cell cycle. In addition, geminin directly interacts with Six3 and Hox homeodomain proteins during embryogenesis and inhibits their functions. The regulation of Hox function by geminin also involves a transient association with the Hox repressive Polycomb complex. The functions of geminin to obstruct key molecules of both cell proliferation and embryonic development suggest a competitive coordination of these two processes.
Cellular and Molecular Life Sciences CMLS 08/2005; 62(13):1425-33. · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neural development in the chick embryo is now understood in great detail on a cellular and a molecular level. It begins already before gastrulation, when a separation of neural and epidermal cell fates occurs under the control of FGF and BMP/Wnt signalling, respectively. This early specification becomes further refined around the tip of the primitive streak, until finally the anterior-posterior level of the neuroectoderm becomes established through progressive caudalization. In this review we focus on processes in the chick embryo and put classical and more recent molecular data into a coherent scenario.
Mechanisms of Development 10/2004; 121(9):1031-42. · 2.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: As in many other animals, the primordial germ cells (PGCs) in avian and reptile embryos are specified in positions distinct from the positions where they differentiate into sperm and egg. Unlike in other organism however, in these embryos, the PGCs use the vascular system as a vehicle to transport them to the region of the gonad where they exit the blood vessels and reach their target. To determine the molecular mechanisms governing PGC migration in these species, we have investigated the role of the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) in guiding the cells towards their target in the chick embryo. We show that sdf-1 mRNA is expressed in locations where PGCs are found and towards which they migrate at the time they leave the blood vessels. Ectopically expressed chicken SDF-1alpha led to accumulation of PGCs at those positions. This analysis, as well as analysis of gene expression and PGC behavior in the mouse embryo, suggest that in both organisms, SDF-1 functions during the second phase of PGC migration, and not at earlier phases. These findings suggest that SDF-1 is required for the PGCs to execute the final migration steps as they transmigrate through the blood vessel endothelium of the chick or the gut epithelium of the mouse.
[Show abstract][Hide abstract] ABSTRACT: Growth and differentiation are two major themes in embryonic development. Numerous cell divisions have to be regulated on the path from a unicellular embryo, the zygote, to the multicellular structures of a mature being. Numerous functions, specializations and cellular identities have to be generated, in order to form a complex and mature animal. Numerous mechanisms have to control the correct assignment and acquisition of cellular fates, as well as the right timing and allocation of cells. Therefore, a strict coordination has to occur between embryonic patterning and the cell cycle. From this point of view, dual roles or mutual interactions of typical proliferation and developmental control genes are likely. Recently, new light was shed on these issues by identifying the nuclear protein Geminin as a molecular coordinator between the cell cycle and axial patterning. We summarize the role of Geminin in cell cycle, in the embryonic patterning controlled by Hox genes, providing insights into cell cycle regulators in embryonic development, and conversely, typical developmental control genes in cell cycle regulation.
[Show abstract][Hide abstract] ABSTRACT: During early vertebrate development, ANF homeobox genes are expressed in the prospective forebrain. Their regulation is essential for correct morphogenesis and function of the prosencephalon. We identified a 1-kb fragment upstream of the chicken GANF gene sufficient to drive lacZ expression in the endogenous expression domain. Concordant with the high conservation of this sequence in five investigated species, this element is also active in the corresponding expression domain of the zebrafish orthologue. In vivo analysis of two in vitro-identified Otx2 binding sites in this conserved sequence revealed their necessity for activation of the chicken ANF promoter. In addition, we identified a Pax6-binding site close to the transcriptional start site that is occupied in vivo by Pax6 protein. Pax6 and GANF exhibit mutually exclusive expression domains in the anterior embryonic region. Overexpression of Pax6 in chick embryos inhibited the endogenous GANF expression, and in Pax6(-/-) mice the expression domain of the murine ANF orthologue Hesx1 was expanded and sustained, indicating inhibitory effects of Pax6 on GANF. However, a mutation of the Pax6 site did not abolish reporter activity from an electroporated vector. We conclude that Otx2 and Pax6 are key molecules involved in conserved mechanisms of ANF gene regulation.
[Show abstract][Hide abstract] ABSTRACT: Ezrin is a member of the ERM- (Ezrin-Radixin-Moesin-) family of actin binding proteins, which function as linkers of the cortical cytoskeleton to components of the plasma membrane. Additional roles for Ezrin in intracellular signalling and ion channel regulation were suggested. We found Ezrin mRNA in the anterior endo- and mesoderm of chick gastrula stage embryos. In these tissues Ezrin message is strongly expressed throughout early development of the foregut (pharynx) and heart tube. During later stages of development, highly restricted expression domains of Ezrin mRNA were detected in the endodermal lining of the pharyngeal pouches, the mesonephric duct and tubuli, and in the ectodermal placodes giving rise to the inner ear, eye lens and olfactory epithelium.
[Show abstract][Hide abstract] ABSTRACT: Embryonic development is tightly controlled. The clustered genes of the Hox family of homeobox proteins play an important part in regulating this development and also proliferation. They specify embryonic structures along the body axis, and are associated with normal and malignant cell growth. The cell-cycle regulator geminin controls replication by binding to the licensing factor Cdt1, and is involved in neural differentiation. Here, we show that murine geminin associates transiently with members of the Hox-repressing polycomb complex, with the chromatin of Hox regulatory DNA elements and with Hox proteins. Gain- and loss-of-function experiments in the chick neural tube demonstrate that geminin modulates the anterior boundary of Hoxb9 transcription, which suggests a polycomb-like activity for geminin. The interaction between geminin and Hox proteins prevents Hox proteins from binding to DNA, inhibits Hox-dependent transcriptional activation of reporter and endogenous downstream target genes, and displaces Cdt1 from its complex with geminin. By establishing competitive regulation, geminin functions as a coordinator of developmental and proliferative control.
[Show abstract][Hide abstract] ABSTRACT: Bone morphogenetic proteins (BMPs) control the development of diverse tissues during embryogenesis. Here, we report the expression of the BMP10 gene during chick development. BMP10 transcription is confined to the myocardium of atriae and ventricles in the forming heart from day 2 of development onwards (stage HH13). It correlates with the thickening of the innermost layer of the walls, i.e. the formation of myocardial ridges or trabeculae.
Development Genes and Evolution 03/2004; 214(2):96-8. · 1.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The development of avian embryos is characterized by the large amount of yolk present from the one-cell stage until late phases of organogenesis. In the chick, an axis of bilateral symmetry is established already before egg laying, when the egg rotates in the uterus. There is evidence for an active Wnt-catenin pathway in the vegetal cells in the periphery of the multi-cellular embryo. It overlaps with the posteriorly restricted expression of genes characterizing the vegetal hemisphere in amphibia. The zone of overlap bears several functional characteristics of a Nieuwkoop center, which is first apparent in the posterior marginal zone, but continues into the early primitive streak. Only the anterior part of the late streak is capable of direct neural induction, and only its tip, Hensen's node, can induce an anterior neural identity. This latter activity leaves the node together with the cells representing the anterior mesendoderm. Thus, although the constraints and dynamics of avian development make comparisons with the amphibian situation a complex undertaking, Hensen's node comes as close as possible to an organizer in Spemann and H. Mangold's definition.
The International Journal of Developmental Biology 02/2001; 45(1):281-7. · 2.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The organizer of vertebrate embryos represents the major regulatory center for the formation of the embryonic axis during gastrulation. The early blastopore lip of amphibia and Hensen's node of the chick at the full-length primitive streak stage possess both a head- and a trunk-inducing potential. In mice, a head-inducing activity was identified in the extraembryonic, anterior visceral endoderm (AVE) by tissue ablation and genetic experiments. Evidence for a similar activity in the AVE from the rabbit was obtained by transplanting below the avian epiblast. However, it was still unclear whether the AVE is the exclusive origin of anterior neural induction or if this activity is recapitulated by the node and/or its derivatives. We report here that nodes from both rabbit and mouse embryos can induce a complete neural axis including forebrain structures upon grafting to chick hosts. Thus, in rabbits and mice not only the AVE, but also the node, possesses a potential for the induction of anterior neural tissue.
[Show abstract][Hide abstract] ABSTRACT: In Drosophila, the tinman homeobox gene is absolutely required for heart development. In the vertebrates, a small family of tinman-related genes, the cardiac NK-2 genes, appear to play a similar role in the formation of the vertebrate heart. However, targeted gene ablation of one of these genes, Nkx2-5, results in defects in only the late stages of cardiac development suggesting the presence of a rescuing gene function early in development. Here, we report the characterization of a novel tinman-related gene, XNkx2-10, which is expressed during early heart development in Xenopus. Using in vitro assays, we show that XNkx2-10 is capable of transactivating expression from promoters previously shown to be activated by other tinman-related genes, including Nkx2-5. Furthermore, Xenopus Nkx2-10 can synergize with the GATA-4 and SRF transcription factors to activate reporter gene expression.
Mechanisms of Development 04/2000; 91(1-2):369-73. · 2.38 Impact Factor