Development

The ATP-dependent chromatin remodeling enzyme CHD7 regulates pro-neural gene expression and neurogenesis in the inner ear.

Publication Date: 2010 Sep PMID: 20736290
Authors: Hurd, E. A. - Poucher, H. K. - Cheng, K. - Raphael, Y. - Martin, D. M.
Journal: Development

Inner ear neurogenesis is positively regulated by the pro-neural bHLH transcription factors Ngn1 and NeuroD, but the factors that act upstream of this regulation are not well understood. Recent evidence in mouse and Drosophila suggests that neural development depends on proper chromatin remodeling, both for maintenance of neural stem cells and for proper neuronal differentiation. Here, we show that CHD7, an ATP-dependent chromatin remodeling enzyme mutated in human CHARGE syndrome, is necessary for proliferation of inner ear neuroblasts and inner ear morphogenesis. Conditional deletion of Chd7 in the developing otocyst using Foxg1-Cre resulted in cochlear hypoplasia and complete absence of the semicircular canals and cristae. Conditional knockout and null otocysts also had reductions in vestibulo-cochlear ganglion size and neuron number in combination with reduced expression of Ngn1, Otx2 and Fgf10, concurrent with expansion of the neural fate suppressor Tbx1 and reduced cellular proliferation. Heterozygosity for Chd7 mutations had no major effects on expression of otic patterning genes or on cell survival, but resulted in decreased proliferation within the neurogenic domain. These data indicate that epigenetic regulation of gene expression by CHD7 must be tightly coordinated for proper development of inner ear neuroblasts.

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The EGR family gene egrh-1 functions non-autonomously in the control of oocyte meiotic maturation and ovulation in C. elegans.

Publication Date: 2010 Sep PMID: 20736289
Authors: Clary, L. M. - Okkema, P. G.
Journal: Development

Oocyte production, maturation and ovulation must be coordinated with sperm availability for successful fertilization. In C. elegans this coordination involves signals from the sperm to the oocyte and somatic gonad, which stimulate maturation and ovulation. We have found that the C. elegans early growth response factor family member EGRH-1 inhibits oocyte maturation and ovulation until sperm are available. In the absence of sperm, egrh-1 mutants exhibit derepressed oocyte maturation marked by MAPK activation and ovulation. egrh-1 mutants exhibit ectopic oocyte differentiation in the distal gonadal arm and accumulate abnormal and degraded oocytes proximally. These defects result in reduced brood size and partially penetrant embryonic lethality. We have found that endogenous EGRH-1 protein and an egrh-1::gfp reporter gene are expressed in the sheath and distal tip cells of the somatic gonad, the gut and other non-gonadal tissues, as well as in sperm, but expression is not observed in oocytes. Results of tissue-specific egrh-1(RNAi) experiments and genetic mosaic analyses revealed that EGRH-1 function is necessary in the soma and, surprisingly, this function is required in both the gut and the somatic gonad. Based on transformation rescue experiments we hypothesize that EGRH-1 in the somatic gonad inhibits oocyte maturation and ovulation.

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Moesin1 and Ve-cadherin are required in endothelial cells during in vivo tubulogenesis.

Publication Date: 2010 Sep PMID: 20736288
Authors: Wang, Y. - Kaiser, M. S. - Larson, J. D. - Nasevicius, A. - Clark, K. J. - Wadman, S. A. - Roberg-Perez, S. E. - Ekker, S. C. - Hackett, P. B. - McGrail, M. - Essner, J. J.
Journal: Development

Endothelial tubulogenesis is a crucial step in the formation of functional blood vessels during angiogenesis and vasculogenesis. Here, we use in vivo imaging of living zebrafish embryos expressing fluorescent fusion proteins of beta-Actin, alpha-Catenin, and the ERM family member Moesin1 (Moesin a), to define a novel cord hollowing process that occurs during the initial stages of tubulogenesis in intersegmental vessels (ISVs) in the embryo. We show that the primary lumen elongates along cell junctions between at least two endothelial cells during embryonic angiogenesis. Moesin1-EGFP is enriched around structures that resemble intracellular vacuoles, which fuse with the luminal membrane during expansion of the primary lumen. Analysis of silent heart mutant embryos shows that initial lumen formation in the ISVs is not dependent on blood flow; however, stabilization of a newly formed lumen is dependent upon blood flow. Zebrafish moesin1 knockdown and cell transplantation experiments demonstrate that Moesin1 is required in the endothelial cells of the ISVs for in vivo lumen formation. Our analyses suggest that Moesin1 contributes to the maintenance of apical/basal cell polarity of the ISVs as defined by adherens junctions. Knockdown of the adherens junction protein Ve-cadherin disrupts formation of the apical membrane and lumen in a cell-autonomous manner. We suggest that Ve-cadherin and Moesin1 function to establish and maintain apical/basal polarity during multicellular lumen formation in the ISVs.

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HLH54F is required for the specification and migration of longitudinal gut muscle founders from the caudal mesoderm of Drosophila.

Publication Date: 2010 Sep PMID: 20736287
Authors: Ismat, A. - Schaub, C. - Reim, I. - Kirchner, K. - Schultheis, D. - Frasch, M.
Journal: Development

HLH54F, the Drosophila ortholog of the vertebrate basic helix-loop-helix domain-encoding genes capsulin and musculin, is expressed in the founder cells and developing muscle fibers of the longitudinal midgut muscles. These cells descend from the posterior-most portion of the mesoderm, termed the caudal visceral mesoderm (CVM), and migrate onto the trunk visceral mesoderm prior to undergoing myoblast fusion and muscle fiber formation. We show that HLH54F expression in the CVM is regulated by a combination of terminal patterning genes and snail. We generated HLH54F mutations and show that this gene is crucial for the specification, migration and survival of the CVM cells and the longitudinal midgut muscle founders. HLH54F mutant embryos, larvae, and adults lack all longitudinal midgut muscles, which causes defects in gut morphology and integrity. The function of HLH54F as a direct activator of gene expression is exemplified by our analysis of a CVM-specific enhancer from the Dorsocross locus, which requires combined inputs from HLH54F and Biniou in a feed-forward fashion. We conclude that HLH54F is the earliest specific regulator of CVM development and that it plays a pivotal role in all major aspects of development and differentiation of this largely twist-independent population of mesodermal cells.

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Axolotl Nanog activity in mouse embryonic stem cells demonstrates that ground state pluripotency is conserved from urodele amphibians to mammals.

Publication Date: 2010 Sep PMID: 20736286
Authors: Dixon, J. E. - Allegrucci, C. - Redwood, C. - Kump, K. - Bian, Y. - Chatfield, J. - Chen, Y. H. - Sottile, V. - Voss, S. R. - Alberio, R. - Johnson, A. D.
Journal: Development

Cells in the pluripotent ground state can give rise to somatic cells and germ cells, and the acquisition of pluripotency is dependent on the expression of Nanog. Pluripotency is conserved in the primitive ectoderm of embryos from mammals and urodele amphibians, and here we report the isolation of a Nanog ortholog from axolotls (axNanog). axNanog does not contain a tryptophan repeat domain and is expressed as a monomer in the axolotl animal cap. The monomeric form is sufficient to regulate pluripotency in mouse embryonic stem cells, but axNanog dimers are required to rescue LIF-independent self-renewal. Our results show that protein interactions mediated by Nanog dimerization promote proliferation. More importantly, they demonstrate that the mechanisms governing pluripotency are conserved from urodele amphibians to mammals.

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Recognition of pre- and postsynaptic neurons via nephrin/NEPH1 homologs is a basis for the formation of the Drosophila retinotopic map.

Publication Date: 2010 Aug 19 PMID: 20724453
Authors: Sugie, A. - Umetsu, D. - Yasugi, T. - Fischbach, K. F. - Tabata, T.
Journal: Development

Topographic maps, which maintain the spatial order of neurons in the order of their axonal connections, are found in many parts of the nervous system. Here, we focus on the communication between retinal axons and their postsynaptic partners, lamina neurons, in the first ganglion of the Drosophila visual system, as a model for the formation of topographic maps. Post-mitotic lamina precursor cells differentiate upon receiving Hedgehog signals delivered through newly arriving retinal axons and, before maturing to extend neurites, extend short processes toward retinal axons to create the lamina column. The lamina column provides the cellular basis for establishing stereotypic synapses between retinal axons and lamina neurons. In this study, we identified two cell-adhesion molecules: Hibris, which is expressed in post-mitotic lamina precursor cells; and Roughest, which is expressed on retinal axons. Both proteins belong to the nephrin/NEPH1 family. We provide evidence that recognition between post-mitotic lamina precursor cells and retinal axons is mediated by interactions between Hibris and Roughest. These findings revealed mechanisms by which axons of presynaptic neurons deliver signals to induce the development of postsynaptic partners at the target area. Postsynaptic partners then recognize the presynaptic axons to make ensembles, thus establishing a topographic map along the anterior/posterior axis.

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miR-18, a member of Oncomir-1, targets heat shock transcription factor 2 in spermatogenesis.

Publication Date: 2010 Aug 19 PMID: 20724452
Authors: Bjork, J. K. - Sandqvist, A. - Elsing, A. N. - Kotaja, N. - Sistonen, L.
Journal: Development

miR-18 belongs to the Oncomir-1 or miR-17 approximately 92 cluster that is intimately associated with the occurrence and progression of different types of cancer. However, the physiological roles of the Oncomir-1 cluster and its individual miRNAs are largely unknown. Here, we describe a novel function for miR-18 in mouse. We show that miR-18 directly targets heat shock factor 2 (HSF2), a transcription factor that influences a wide range of developmental processes including embryogenesis and gametogenesis. Furthermore, we show that miR-18 is highly abundant in testis, displaying distinct cell-type-specific expression during the epithelial cycle that constitutes spermatogenesis. Expression of HSF2 and of miR-18 exhibit an inverse correlation during spermatogenesis, indicating that, in germ cells, HSF2 is downregulated by miR-18. To investigate the in vivo function of miR-18 we developed a novel method, T-GIST, and demonstrate that inhibition of miR-18 in intact seminiferous tubules leads to increased HSF2 protein levels and altered expression of HSF2 target genes. Our results reveal that miR-18 regulates HSF2 activity in spermatogenesis and link miR-18 to HSF2-mediated physiological processes such as male germ cell maturation.

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Drosophila Ataxin 2-binding protein 1 marks an intermediate step in the molecular differentiation of female germline cysts.

Publication Date: 2010 Aug 19 PMID: 20724451
Authors: Tastan, O. Y. - Maines, J. Z. - Li, Y. - McKearin, D. M. - Buszczak, M.
Journal: Development

In the Drosophila ovary, extrinsic signaling from the niche and intrinsic translational control machinery regulate the balance between germline stem cell maintenance and the differentiation of their daughters. However, the molecules that promote the continued stepwise development of ovarian germ cells after their exit from the niche remain largely unknown. Here, we report that the early development of germline cysts depends on the Drosophila homolog of the human ataxin 2-binding protein 1 (A2BP1) gene. Drosophila A2BP1 protein expression is first observed in the cytoplasm of 4-, 8- and 16-cell cysts, bridging the expression of the early differentiation factor Bam with late markers such as Orb, Rbp9 and Bruno encoded by arrest. The expression of A2BP1 is lost in bam, sans-fille (snf) and mei-P26 mutants, but is still present in other mutants such as rbp9 and arrest. A2BP1 alleles of varying strength produce mutant phenotypes that include germline counting defects and cystic tumors. Phenotypic analysis reveals that strong A2BP1 alleles disrupt the transition from mitosis to meiosis. These mutant cells continue to express high levels of mitotic cyclins and fail to express markers of terminal differentiation. Biochemical analysis reveals that A2BP1 isoforms bind to each other and associate with Bruno, a known translational repressor protein. These data show that A2BP1 promotes the molecular differentiation of ovarian germline cysts.

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Hand2 ensures an appropriate environment for cardiac fusion by limiting Fibronectin function.

Publication Date: 2010 Aug 19 PMID: 20724450
Authors: Garavito-Aguilar, Z. V. - Riley, H. E. - Yelon, D.
Journal: Development

Heart formation requires the fusion of bilateral cardiomyocyte populations as they move towards the embryonic midline. The bHLH transcription factor Hand2 is essential for cardiac fusion; however, the effector genes that execute this function of Hand2 are unknown. Here, we provide in zebrafish the first evidence for a downstream component of the Hand2 pathway that mediates cardiac morphogenesis. Although hand2 is expressed in cardiomyocytes, mosaic analysis demonstrates that it plays a non-autonomous role in regulating cardiomyocyte movement. Gene expression profiles reveal heightened expression of fibronectin 1 (fn1) in hand2 mutant embryos. Reciprocally, overexpression of hand2 leads to decreased Fibronectin levels. Furthermore, reduction of fn1 function enables rescue of cardiac fusion in hand2 mutants: bilateral cardiomyocyte populations merge and exhibit improved tissue architecture, albeit without major changes in apicobasal polarity. Together, our data provide a novel example of a tissue creating a favorable environment for its morphogenesis: the Hand2 pathway establishes an appropriate environment for cardiac fusion through negative modulation of Fn1 levels.

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Inhibition of Wnt signaling by Wise (Sostdc1) and negative feedback from Shh controls tooth number and patterning.

Publication Date: 2010 Aug 19 PMID: 20724449
Authors: Ahn, Y. - Sanderson, B. W. - Klein, O. D. - Krumlauf, R.
Journal: Development

Mice carrying mutations in Wise (Sostdc1) display defects in many aspects of tooth development, including tooth number, size and cusp pattern. To understand the basis of these defects, we have investigated the pathways modulated by Wise in tooth development. We present evidence that, in tooth development, Wise suppresses survival of the diastema or incisor vestigial buds by serving as an inhibitor of Lrp5- and Lrp6-dependent Wnt signaling. Reducing the dosage of the Wnt co-receptor genes Lrp5 and Lrp6 rescues the Wise-null tooth phenotypes. Inactivation of Wise leads to elevated Wnt signaling and, as a consequence, vestigial tooth buds in the normally toothless diastema region display increased proliferation and continuous development to form supernumerary teeth. Conversely, gain-of-function studies show that ectopic Wise reduces Wnt signaling and tooth number. Our analyses demonstrate that the Fgf and Shh pathways are major downstream targets of Wise-regulated Wnt signaling. Furthermore, our experiments revealed that Shh acts as a negative-feedback regulator of Wnt signaling and thus determines the fate of the vestigial buds and later tooth patterning. These data provide insight into the mechanisms that control Wnt signaling in tooth development and into how crosstalk among signaling pathways controls tooth number and morphogenesis.

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The ubiquitin proteasome system is required for cell proliferation of the lens epithelium and for differentiation of lens fiber cells in zebrafish.

Publication Date: 2010 Aug 19 PMID: 20724448
Authors: Imai, F. - Yoshizawa, A. - Fujimori-Tonou, N. - Kawakami, K. - Masai, I.
Journal: Development

In the developing vertebrate lens, epithelial cells differentiate into fiber cells, which are elongated and flat in shape and form a multilayered lens fiber core. In this study, we identified the zebrafish volvox (vov) mutant, which shows defects in lens fiber differentiation. In the vov mutant, lens epithelial cells fail to proliferate properly. Furthermore, differentiating lens fiber cells do not fully elongate, and the shape and position of lens fiber nuclei are affected. We found that the vov mutant gene encodes Psmd6, the subunit of the 26S proteasome. The proteasome regulates diverse cellular functions by degrading polyubiquitylated proteins. Polyubiquitylated proteins accumulate in the vov mutant. Furthermore, polyubiquitylation is active in nuclei of differentiating lens fiber cells, suggesting roles of the proteasome in lens fiber differentiation. We found that an E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) is involved in lens defects in the vov mutant. These data suggest that the ubiquitin proteasome system is required for cell proliferation of lens epithelium and for the differentiation of lens fiber cells in zebrafish.

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Mouse Emi2 as a distinctive regulatory hub in second meiotic metaphase.

Publication Date: 2010 Aug 19 PMID: 20724447
Authors: Suzuki, T. - Suzuki, E. - Yoshida, N. - Kubo, A. - Li, H. - Okuda, E. - Amanai, M. - Perry, A. C.
Journal: Development

The oocytes of vertebrates are typically arrested at metaphase II (mII) by the cytostatic factor Emi2 until fertilization. Regulatory mechanisms in Xenopus Emi2 (xEmi2) are understood in detail but contrastingly little is known about the corresponding mechanisms in mammals. Here, we analyze Emi2 and its regulatory neighbours at the molecular level in intact mouse oocytes. Emi2, but not xEmi2, exhibited nuclear targeting. Unlike xEmi2, separable N- and C-terminal domains of mouse Emi2 modulated metaphase establishment and maintenance, respectively, through indirect and direct mechanisms. The C-terminal activity was mapped to the potential phosphorylation target Tx(5)SxS, a destruction box (D-box), a lattice of Zn(2+)-coordinating residues and an RL domain. The minimal region of Emi2 required for its cytostatic activity was mapped to a region containing these motifs, from residue 491 to the C terminus. The cytostatic factor Mos-MAPK promoted Emi2-dependent metaphase establishment, but Mos autonomously disappeared from meiotically competent mII oocytes. The N-terminal Plx1-interacting phosphodegron of xEmi2 was apparently shifted to within a minimal fragment (residues 51-300) of mouse Emi2 that also contained a calmodulin kinase II (CaMKII) phosphorylation motif and which was efficiently degraded during mII exit. Two equimolar CaMKII gamma isoform variants were present in mII oocytes, neither of which phosphorylated Emi2 in vitro, consistent with the involvement of additional factors. No evidence was found that calcineurin is required for mouse mII exit. These data support a model in which mammalian meiotic establishment, maintenance and exit converge upon a modular Emi2 hub via evolutionarily conserved and divergent mechanisms.

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Coordinated sequential action of EGFR and Notch signaling pathways regulates proneural wave progression in the Drosophila optic lobe.

Publication Date: 2010 Aug 19 PMID: 20724446
Authors: Yasugi, T. - Sugie, A. - Umetsu, D. - Tabata, T.
Journal: Development

During neurogenesis in the medulla of the Drosophila optic lobe, neuroepithelial cells are programmed to differentiate into neuroblasts at the medial edge of the developing optic lobe. The wave of differentiation progresses synchronously in a row of cells from medial to the lateral regions of the optic lobe, sweeping across the entire neuroepithelial sheet; it is preceded by the transient expression of the proneural gene lethal of scute [l(1)sc] and is thus called the proneural wave. We found that the epidermal growth factor receptor (EGFR) signaling pathway promotes proneural wave progression. EGFR signaling is activated in neuroepithelial cells and induces l(1)sc expression. EGFR activation is regulated by transient expression of Rhomboid (Rho), which is required for the maturation of the EGF ligand Spitz. Rho expression is also regulated by the EGFR signal. The transient and spatially restricted expression of Rho generates sequential activation of EGFR signaling and assures the directional progression of the differentiation wave. This study also provides new insights into the role of Notch signaling. Expression of the Notch ligand Delta is induced by EGFR, and Notch signaling prolongs the proneural state. Notch signaling activity is downregulated by its own feedback mechanism that permits cells at proneural states to subsequently develop into neuroblasts. Thus, coordinated sequential action of the EGFR and Notch signaling pathways causes the proneural wave to progress and induce neuroblast formation in a precisely ordered manner.

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An SNP in an ultraconserved regulatory element affects Dlx5/Dlx6 regulation in the forebrain.

Publication Date: 2010 Sep PMID: 20702565
Authors: Poitras, L. - Yu, M. - Lesage-Pelletier, C. - Macdonald, R. B. - Gagne, J. P. - Hatch, G. - Kelly, I. - Hamilton, S. P. - Rubenstein, J. L. - Poirier, G. G. - Ekker, M.
Journal: Development

Dlx homeobox genes play a crucial role in the migration and differentiation of the subpallial precursor cells that give rise to various subtypes of gamma-aminobutyric acid (GABA)-expressing neurons of the forebrain, including local-circuit cortical interneurons. Aberrant development of GABAergic interneurons has been linked to several neurodevelopmental disorders, including epilepsy, schizophrenia, Rett syndrome and autism. Here, we report in mice that a single-nucleotide polymorphism (SNP) found in an autistic proband falls within a functional protein binding site in an ultraconserved cis-regulatory element. This element, I56i, is involved in regulating Dlx5/Dlx6 homeobox gene expression in the developing forebrain. We show that the SNP results in reduced I56i activity, predominantly in the medial and caudal ganglionic eminences and in streams of neurons tangentially migrating to the cortex. Reduced activity is also observed in GABAergic interneurons of the adult somatosensory cortex. The SNP affects the affinity of Dlx proteins for their binding site in vitro and reduces the transcriptional activation of the enhancer by Dlx proteins. Affinity purification using I56i sequences led to the identification of a novel regulator of Dlx gene expression, general transcription factor 2 I (Gtf2i), which is among the genes most often deleted in Williams-Beuren syndrome, a neurodevelopmental disorder. This study illustrates the clear functional consequences of a single nucleotide variation in an ultraconserved non-coding sequence in the context of developmental abnormalities associated with disease.

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Sall1-dependent signals affect Wnt signaling and ureter tip fate to initiate kidney development.

Publication Date: 2010 Sep PMID: 20702564
Authors: Kiefer, S. M. - Robbins, L. - Stumpff, K. M. - Lin, C. - Ma, L. - Rauchman, M.
Journal: Development

Development of the metanephric kidney depends on precise control of branching of the ureteric bud. Branching events represent terminal bifurcations that are thought to depend on unique patterns of gene expression in the tip compared with the stalk and are influenced by mesenchymal signals. The metanephric mesenchyme-derived signals that control gene expression at the ureteric bud tip are not well understood. In mouse Sall1 mutants, the ureteric bud grows out and invades the metanephric mesenchyme, but it fails to initiate branching despite tip-specific expression of Ret and Wnt11. The stalk-specific marker Wnt9b and the beta-catenin downstream target Axin2 are ectopically expressed in the mutant ureteric bud tips, suggesting that upregulated canonical Wnt signaling disrupts ureter branching in this mutant. In support of this hypothesis, ureter arrest is rescued by lowering beta-catenin levels in the Sall1 mutant and is phenocopied by ectopic expression of a stabilized beta-catenin in the ureteric bud. Furthermore, transgenic overexpression of Wnt9b in the ureteric bud causes reduced branching in multiple founder lines. These studies indicate that Sall1-dependent signals from the metanephric mesenchyme are required to modulate ureteric bud tip Wnt patterning in order to initiate branching.

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The role of Drosophila Lamin C in muscle function and gene expression.

Publication Date: 2010 Sep PMID: 20702563
Authors: Dialynas, G. - Speese, S. - Budnik, V. - Geyer, P. K. - Wallrath, L. L.
Journal: Development

The inner side of the nuclear envelope (NE) is lined with lamins, a meshwork of intermediate filaments that provides structural support for the nucleus and plays roles in many nuclear processes. Lamins, classified as A- or B-types on the basis of biochemical properties, have a conserved globular head, central rod and C-terminal domain that includes an Ig-fold structural motif. In humans, mutations in A-type lamins give rise to diseases that exhibit tissue-specific defects, such as Emery-Dreifuss muscular dystrophy. Drosophila is being used as a model to determine tissue-specific functions of A-type lamins in development, with implications for understanding human disease mechanisms. The GAL4-UAS system was used to express wild-type and mutant forms of Lamin C (the presumed Drosophila A-type lamin), in an otherwise wild-type background. Larval muscle-specific expression of wild type Drosophila Lamin C caused no overt phenotype. By contrast, larval muscle-specific expression of a truncated form of Lamin C lacking the N-terminal head (Lamin C DeltaN) caused muscle defects and semi-lethality, with adult 'escapers' possessing malformed legs. The leg defects were due to a lack of larval muscle function and alterations in hormone-regulated gene expression. The consequences of Lamin C association at a gene were tested directly by targeting a Lamin C DNA-binding domain fusion protein upstream of a reporter gene. Association of Lamin C correlated with localization of the reporter gene at the nuclear periphery and gene repression. These data demonstrate connections among the Drosophila A-type lamin, hormone-induced gene expression and muscle function.

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The iron exporter ferroportin 1 is essential for development of the mouse embryo, forebrain patterning and neural tube closure.

Publication Date: 2010 Sep PMID: 20702562
Authors: Mao, J. - McKean, D. M. - Warrier, S. - Corbin, J. G. - Niswander, L. - Zohn, I. E.
Journal: Development

Neural tube defects (NTDs) are some of the most common birth defects observed in humans. The incidence of NTDs can be reduced by peri-conceptional folic acid supplementation alone and reduced even further by supplementation with folic acid plus a multivitamin. Here, we present evidence that iron maybe an important nutrient necessary for normal development of the neural tube. Following implantation of the mouse embryo, ferroportin 1 (Fpn1) is essential for the transport of iron from the mother to the fetus and is expressed in the visceral endoderm, yolk sac and placenta. The flatiron (ffe) mutant mouse line harbors a hypomorphic mutation in Fpn1 and we have created an allelic series of Fpn1 mutations that result in graded developmental defects. A null mutation in the Fpn1 gene is embryonic lethal before gastrulation, hypomorphic Fpn1(ffe/ffe) mutants exhibit NTDs consisting of exencephaly, spina bifida and forebrain truncations, while Fpn1(ffe/KI) mutants exhibit even more severe NTDs. We show that Fpn1 is not required in the embryo proper but rather in the extra-embryonic visceral endoderm. Our data indicate that loss of Fpn1 results in abnormal morphogenesis of the anterior visceral endoderm (AVE). Defects in the development of the forebrain in Fpn1 mutants are compounded by defects in multiple signaling centers required for maintenance of the forebrain, including the anterior definitive endoderm (ADE), anterior mesendoderm (AME) and anterior neural ridge (ANR). Finally, we demonstrate that this loss of forebrain maintenance is due in part to the iron deficiency that results from the absence of fully functional Fpn1.

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Arterial pole progenitors interpret opposing FGF/BMP signals to proliferate or differentiate.

Publication Date: 2010 Sep PMID: 20702561
Authors: Hutson, M. R. - Zeng, X. L. - Kim, A. J. - Antoon, E. - Harward, S. - Kirby, M. L.
Journal: Development

During heart development, a subpopulation of cells in the heart field maintains cardiac potential over several days of development and forms the myocardium and smooth muscle of the arterial pole. Using clonal and explant culture experiments, we show that these cells are a stem cell population that can differentiate into myocardium, smooth muscle and endothelial cells. The multipotent stem cells proliferate or differentiate into different cardiovascular cell fates through activation or inhibition of FGF and BMP signaling pathways. BMP promoted myocardial differentiation but not proliferation. FGF signaling promoted proliferation and induced smooth muscle differentiation, but inhibited myocardial differentiation. Blocking the Ras/Erk intracellular pathway promoted myocardial differentiation, while the PLCgamma and PI3K pathways regulated proliferation. In vivo, inhibition of both pathways resulted in predictable arterial pole defects. These studies suggest that myocardial differentiation of arterial pole progenitors requires BMP signaling combined with downregulation of the FGF/Ras/Erk pathway. The FGF pathway maintains the pool of proliferating stem cells and later promotes smooth muscle differentiation.

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BMP-mediated inhibition of FGF signaling promotes cardiomyocyte differentiation of anterior heart field progenitors.

Publication Date: 2010 Sep PMID: 20702560
Authors: Tirosh-Finkel, L. - Zeisel, A. - Brodt-Ivenshitz, M. - Shamai, A. - Yao, Z. - Seger, R. - Domany, E. - Tzahor, E.
Journal: Development

The anterior heart field (AHF) encompasses a niche in which mesoderm-derived cardiac progenitors maintain their multipotent and undifferentiated nature in response to signals from surrounding tissues. Here, we investigate the signaling mechanism that promotes the shift from proliferating cardiac progenitors to differentiating cardiomyocytes in chick embryos. Genomic and systems biology approaches, as well as perturbations of signaling molecules, in vitro and in vivo, reveal tight crosstalk between the bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) signaling pathways within the AHF niche: BMP4 promotes myofibrillar gene expression and cardiomyocyte contraction by blocking FGF signaling. Furthermore, inhibition of the FGF-ERK pathway is both sufficient and necessary for these processes, suggesting that FGF signaling blocks premature differentiation of cardiac progenitors in the AHF. We further revealed that BMP4 induced a set of neural crest-related genes, including MSX1. Overexpression of Msx1 was sufficient to repress FGF gene expression and cell proliferation, thereby promoting cardiomyocyte differentiation. Finally, we show that BMP-induced cardiomyocyte differentiation is diminished following cranial neural crest ablation, underscoring the key roles of these cells in the regulation of AHF cell differentiation. Hence, BMP and FGF signaling pathways act via inter- and intra-regulatory loops in multiple tissues, to coordinate the balance between proliferation and differentiation of cardiac progenitors.

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Experimental evidence for the ectodermal origin of the epithelial anlage of the chicken bursa of Fabricius.

Publication Date: 2010 Sep PMID: 20702559
Authors: Nagy, N. - Olah, I.
Journal: Development

The bursa of Fabricius (BF) is a central lymphoid organ of birds responsible for B-cell maturation within bursal follicles of epithelial origin. Despite the fundamental importance of the BF to the birth of B lymphocytes in the immune system, the embryological origin of the epithelial component of the BF remains unknown. The BF arises in the tail bud, caudal to the cloaca and in close association with the cloacal membrane, where the anal invagination (anal sinus) of ectoderm and the caudal endodermal wall of the cloaca are juxtaposed. Serial semi-thin sections of the tail bud show that the anal sinus gradually transforms into the bursal duct and proctodeum, which joins the distal part of the cloaca during late embryogenesis. These anatomical findings raise the possibility that the ectoderm may contribute to the epithelial anlage of the BF. The expression of sonic hedgehog and its receptor in the embryonic gut, but not in the BF, further supports an ectodermal origin for the bursal rudiment. Using chick-quail chimeras, quail tail bud ectoderm was homotopically transplanted into ectoderm-ablated chick, resulting in quail-derived bursal follicle formation. Chimeric bursal anlagen were generated in vitro by recombining chick bursal mesenchyme with quail ectoderm or endoderm and grafting the recombination into the chick coelomic cavity. After hematopoietic cell colonization, bursal follicles formed only in grafts containing BF mesenchyme and tail bud ectoderm. These results strongly support the central role of the ectoderm in the development of the bursal epithelium and hence in the maturation of B lymphocytes.

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The occipital lateral plate mesoderm is a novel source for vertebrate neck musculature.

Publication Date: 2010 Sep 1 PMID: 20699298
Authors: Theis, S. - Patel, K. - Valasek, P. - Otto, A. - Pu, Q. - Harel, I. - Tzahor, E. - Tajbakhsh, S. - Christ, B. - Huang, R.
Journal: Development

In vertebrates, body musculature originates from somites, whereas head muscles originate from the cranial mesoderm. Neck muscles are located in the transition between these regions. We show that the chick occipital lateral plate mesoderm has myogenic capacity and gives rise to large muscles located in the neck and thorax. We present molecular and genetic evidence to show that these muscles not only have a unique origin, but additionally display a distinct temporal development, forming later than any other muscle group described to date. We further report that these muscles, found in the body of the animal, develop like head musculature rather than deploying the programme used by the trunk muscles. Using mouse genetics we reveal that these muscles are formed in trunk muscle mutants but are absent in head muscle mutants. In concordance with this conclusion, their connective tissue is neural crest in origin. Finally, we provide evidence that the mechanism by which these neck muscles develop is conserved in vertebrates.

MeSH Categories: Animals, Animals, Genetically Modified, Avian Proteins/genetics, Chick Embryo, Coturnix, Evolution, Gene Expression Regulation, Developmental, Mesoderm/*embryology, Mice, *Muscle Development/genetics, Mutation, Neck Muscles/*embryology, Neural Crest/embryology, Paired Box Transcription Factors/genetics, Somites/embryology, Transplantation Chimera/embryology/genetics

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The Bicoid gradient is shaped independently of nuclei.

Publication Date: 2010 Sep 1 PMID: 20699297
Authors: Grimm, O. - Wieschaus, E.
Journal: Development

Morphogen gradients provide embryos with positional information, yet how they form is not understood. Binding of the morphogen to receptors could affect the formation of the morphogen gradient, in particular if the number of morphogen binding sites changes with time. For morphogens that function as transcription factors, the final distribution can be heavily influenced by the number of nuclear binding sites. Here, we have addressed the role of the increasing number of nuclei during the formation of the Bicoid gradient in embryos of Drosophila melanogaster. Deletion of a short stretch of sequence in Bicoid impairs its nuclear accumulation. This effect is due to a approximately 4-fold decrease in nuclear import rate and a approximately 2-fold reduction in nuclear residence time compared with the wild-type protein. Surprisingly, the shape of the resulting anterior-posterior gradient as well as the centre-surface distribution are indistinguishable from those of the normal gradient. This suggests that nuclei do not shape the Bicoid gradient but instead function solely during its interpretation.

MeSH Categories: Active Transport, Cell Nucleus, Animals, Animals, Genetically Modified, Body Patterning/genetics/physiology, Cell Nucleus/metabolism, Drosophila melanogaster/embryology/genetics/*metabolism, Genes, Insect, Homeodomain Proteins/genetics/*metabolism, Kinetics, Mutant Proteins/genetics/metabolism, Recombinant Proteins/genetics/metabolism, Sequence Deletion, Trans-Activators/genetics/*metabolism

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The control of axillary meristem fate in the maize ramosa pathway.

Publication Date: 2010 Sep 1 PMID: 20699296
Authors: Gallavotti, A. - Long, J. A. - Stanfield, S. - Yang, X. - Jackson, D. - Vollbrecht, E. - Schmidt, R. J.
Journal: Development

Plant axillary meristems are composed of highly organized, self-renewing stem cells that produce indeterminate branches or terminate in differentiated structures, such as the flowers. These opposite fates, dictated by both genetic and environmental factors, determine interspecific differences in the architecture of plants. The Cys(2)-His(2) zinc-finger transcription factor RAMOSA1 (RA1) regulates the fate of most axillary meristems during the early development of maize inflorescences, the tassel and the ear, and has been implicated in the evolution of grass architecture. Mutations in RA1 or any other known members of the ramosa pathway, RAMOSA2 and RAMOSA3, generate highly branched inflorescences. Here, we report a genetic screen for the enhancement of maize inflorescence branching and the discovery of a new regulator of meristem fate: the RAMOSA1 ENHANCER LOCUS2 (REL2) gene. rel2 mutants dramatically increase the formation of long branches in ears of both ra1 and ra2 mutants. REL2 encodes a transcriptional co-repressor similar to the TOPLESS protein of Arabidopsis, which is known to maintain apical-basal polarity during embryogenesis. REL2 is capable of rescuing the embryonic defects of the Arabidopsis topless-1 mutant, suggesting that REL2 also functions as a transcriptional co-repressor throughout development. We show by genetic and molecular analyses that REL2 physically interacts with RA1, indicating that the REL2/RA1 transcriptional repressor complex antagonizes the formation of indeterminate branches during maize inflorescence development. Our results reveal a novel mechanism for the control of meristem fate and the architecture of plants.

MeSH Categories: Amino Acid Sequence, Arabidopsis Proteins/genetics, Base Sequence, DNA Primase/genetics, Enhancer Elements, Genetic, *Genes, Plant, Hybridization, Genetic, Meristem/growth & development/ultrastructure, Microscopy, Electron, Scanning, Models, Biological, Molecular Sequence Data, Mutagenesis, Phenotype, Plant Proteins/genetics, Protein Interaction Domains and Motifs, Repressor Proteins/genetics, Species Specificity, Transcription Factors/genetics, Zea mays/*genetics/*growth & development/ultrastructure, Zinc Fingers/genetics

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Connecting muscles to tendons: tendons and musculoskeletal development in flies and vertebrates.

Publication Date: 2010 Sep 1 PMID: 20699295
Authors: Schweitzer, R. - Zelzer, E. - Volk, T.
Journal: Development

The formation of the musculoskeletal system represents an intricate process of tissue assembly involving heterotypic inductive interactions between tendons, muscles and cartilage. An essential component of all musculoskeletal systems is the anchoring of the force-generating muscles to the solid support of the organism: the skeleton in vertebrates and the exoskeleton in invertebrates. Here, we discuss recent findings that illuminate musculoskeletal assembly in the vertebrate embryo, findings that emphasize the reciprocal interactions between the forming tendons, muscle and cartilage tissues. We also compare these events with those of the corresponding system in the Drosophila embryo, highlighting distinct and common pathways that promote efficient locomotion while preserving the form of the organism.

MeSH Categories: Animals, Drosophila/embryology/genetics/physiology, Muscles/embryology/physiology, *Musculoskeletal Development/genetics/physiology, Signal Transduction, Tendons/*embryology/physiology, Vertebrates/embryology/genetics/physiology

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An interview with Shin-Ichi Nishikawa. Interview by Kathryn Senior.

Publication Date: 2010 Sep 1 PMID: 20699294
Authors: Nishikawa, S.
Journal: Development

MeSH Categories: Animals, Developmental Biology, Humans, Japan, Research, *Stem Cells

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BMPs and FGFs target Notch signalling via jagged 2 to regulate tooth morphogenesis and cytodifferentiation.

Publication Date: 2010 Sep PMID: 20685737
Authors: Mitsiadis, T. A. - Graf, D. - Luder, H. - Gridley, T. - Bluteau, G.
Journal: Development

The Notch signalling pathway is an evolutionarily conserved intercellular signalling mechanism that is essential for cell fate specification and proper embryonic development. We have analysed the expression, regulation and function of the jagged 2 (Jag2) gene, which encodes a ligand for the Notch family of receptors, in developing mouse teeth. Jag2 is expressed in epithelial cells that give rise to the enamel-producing ameloblasts from the earliest stages of tooth development. Tissue recombination experiments showed that its expression in epithelium is regulated by mesenchyme-derived signals. In dental explants cultured in vitro, the local application of fibroblast growth factors upregulated Jag2 expression, whereas bone morphogenetic proteins provoked the opposite effect. Mice homozygous for a deletion in the Notch-interaction domain of Jag2 presented a variety of severe dental abnormalities. In molars, the crown morphology was misshapen, with additional cusps being formed. This was due to alterations in the enamel knot, an epithelial signalling structure involved in molar crown morphogenesis, in which Bmp4 expression and apoptosis were altered. In incisors, cytodifferentiation and enamel matrix deposition were inhibited. The expression of Tbx1 in ameloblast progenitors, which is a hallmark for ameloblast differentiation and enamel formation, was dramatically reduced in Jag2(-/-) teeth. Together, these results demonstrate that Notch signalling mediated by Jag2 is indispensable for normal tooth development.

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Planar polarity of multiciliated ependymal cells involves the anterior migration of basal bodies regulated by non-muscle myosin II.

Publication Date: 2010 Sep PMID: 20685736
Authors: Hirota, Y. - Meunier, A. - Huang, S. - Shimozawa, T. - Yamada, O. - Kida, Y. S. - Inoue, M. - Ito, T. - Kato, H. - Sakaguchi, M. - Sunabori, T. - Nakaya, M. A. - Nonaka, S. - Ogura, T. - Higuchi, H. - Okano, H. - Spassky, N. - Sawamoto, K.
Journal: Development

Motile cilia generate constant fluid flow over epithelial tissue, and thereby influence diverse physiological processes. Such functions of ciliated cells depend on the planar polarity of the cilia and on their basal bodies being oriented in the downstream direction of fluid flow. Recently, another type of basal body planar polarity, characterized by the anterior localization of the basal bodies in individual cells, was reported in the multiciliated ependymal cells that line the surface of brain ventricles. However, little is known about the cellular and molecular mechanisms by which this polarity is established. Here, we report in mice that basal bodies move in the apical cell membrane during differentiation to accumulate in the anterior region of ependymal cells. The planar cell polarity signaling pathway influences basal body orientation, but not their anterior migration, in the neonatal brain. Moreover, we show by pharmacological and genetic studies that non-muscle myosin II is a key regulator of this distribution of basal bodies. This study demonstrates that the orientation and distribution of basal bodies occur by distinct mechanisms.

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Wnt signaling promotes oral but suppresses aboral structures in Hydractinia metamorphosis and regeneration.

Publication Date: 2010 Sep PMID: 20685735
Authors: Duffy, D. J. - Plickert, G. - Kuenzel, T. - Tilmann, W. - Frank, U.
Journal: Development

We studied the role of Wnt signaling in axis formation during metamorphosis and regeneration in the cnidarian Hydractinia. Activation of Wnt downstream events during metamorphosis resulted in a complete oralization of the animals and repression of aboral structures (i.e. stolons). The expression of Wnt3, Tcf and Brachyury was upregulated and became ubiquitous. Rescue experiments using Tcf RNAi resulted in normal metamorphosis and quantitatively normal Wnt3 and Brachyury expression. Isolated, decapitated polyps regenerated only heads but no stolons. Activation of Wnt downstream targets in regenerating animals resulted in oralization of the polyps. Knocking down Tcf or Wnt3 by RNAi inhibited head regeneration and resulted in complex phenotypes that included ectopic aboral structures. Multiple heads then grew when the RNAi effect had dissipated. Our results provide functional evidence that Wnt promotes head formation but represses the formation of stolons, whereas downregulation of Wnt promotes stolons and represses head formation.

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Notch regulates the switch from symmetric to asymmetric neural stem cell division in the Drosophila optic lobe.

Publication Date: 2010 Sep PMID: 20685734
Authors: Egger, B. - Gold, K. S. - Brand, A. H.
Journal: Development

The proper balance between symmetric and asymmetric stem cell division is crucial both to maintain a population of stem cells and to prevent tumorous overgrowth. Neural stem cells in the Drosophila optic lobe originate within a polarised neuroepithelium, where they divide symmetrically. Neuroepithelial cells are transformed into asymmetrically dividing neuroblasts in a precisely regulated fashion. This cell fate transition is highly reminiscent of the switch from neuroepithelial cells to radial glial cells in the developing mammalian cerebral cortex. To identify the molecules that mediate the transition, we microdissected neuroepithelial cells and compared their transcriptional profile with similarly obtained optic lobe neuroblasts. We find genes encoding members of the Notch pathway expressed in neuroepithelial cells. We show that Notch mutant clones are extruded from the neuroepithelium and undergo premature neurogenesis. A wave of proneural gene expression is thought to regulate the timing of the transition from neuroepithelium to neuroblast. We show that the proneural wave transiently suppresses Notch activity in neuroepithelial cells, and that inhibition of Notch triggers the switch from symmetric, proliferative division, to asymmetric, differentiative division.

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In vivo evidence for transdifferentiation of peripheral neurons.

Publication Date: 2010 Sep PMID: 20685733
Authors: Wright, M. A. - Mo, W. - Nicolson, T. - Ribera, A. B.
Journal: Development

It is commonly thought that differentiated neurons do not give rise to new cells, severely limiting the potential for regeneration and repair of the mature nervous system. However, we have identified cells in zebrafish larvae that first differentiate into dorsal root ganglia sensory neurons but later acquire a sympathetic neuron phenotype. These transdifferentiating neurons are present in wild-type zebrafish. However, they are increased in number in larvae that have a mutant voltage-gated sodium channel gene, scn8aa. Sodium channel knock-down promotes migration of differentiated sensory neurons away from the ganglia. Once in a new environment, sensory neurons transdifferentiate regardless of sodium channel expression. These findings reveal an unsuspected plasticity in differentiated neurons that points to new strategies for treatment of nervous system disease.

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Kctd15 inhibits neural crest formation by attenuating Wnt/{beta}-catenin signaling output.

Publication Date: 2010 Sep PMID: 20685732
Authors: Dutta, S. - Dawid, I. B.
Journal: Development

Neural crest (NC) precursors are stem cells that are capable of forming many cell types after migration to different locations in the embryo. NC and placodes form at the neural plate border (NPB). The Wnt pathway is essential for specifying NC versus placodal identity in this cell population. Here we describe the BTB domain-containing protein Potassium channel tetramerization domain containing 15 (Kctd15) as a factor expressed in the NPB that efficiently inhibits NC induction in zebrafish and frog embryos. Whereas overexpression of Kctd15 inhibited NC formation, knockdown of Kctd15 led to expansion of the NC domain. Likewise, NC induction by Wnt3a plus Chordin in Xenopus animal explants was suppressed by Kctd15, but constitutively active beta-catenin reversed Kctd15-mediated suppression of NC induction. Suppression of NC induction by inhibition of Wnt8.1 was rescued by reduction of Kctd15 expression, linking Kctd15 action to the Wnt pathway. We propose that Kctd15 inhibits NC formation by attenuating the output of the canonical Wnt pathway, thereby restricting expansion of the NC domain beyond its normal range.

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Serotonin 2B receptor signaling is required for craniofacial morphogenesis and jaw joint formation in Xenopus.

Publication Date: 2010 Sep 1 PMID: 20667918
Authors: Reisoli, E. - De Lucchini, S. - Nardi, I. - Ori, M.
Journal: Development

Serotonin (5-HT) is a neuromodulator that plays many different roles in adult and embryonic life. Among the 5-HT receptors, 5-HT2B is one of the key mediators of 5-HT functions during development. We used Xenopus laevis as a model system to further investigate the role of 5-HT2B in embryogenesis, focusing on craniofacial development. By means of gene gain- and loss-of-function approaches and tissue transplantation assays, we demonstrated that 5-HT2B modulates, in a cell-autonomous manner, postmigratory skeletogenic cranial neural crest cell (NCC) behavior without altering early steps of cranial NCC development and migration. 5-HT2B overexpression induced the formation of an ectopic visceral skeletal element and altered the dorsoventral patterning of the branchial arches. Loss-of-function experiments revealed that 5-HT2B signaling is necessary for jaw joint formation and for shaping the mandibular arch skeletal elements. In particular, 5-HT2B signaling is required to define and sustain the Xbap expression necessary for jaw joint formation. To shed light on the molecular identity of the transduction pathway acting downstream of 5-HT2B, we analyzed the function of phospholipase C beta 3 (PLC) in Xenopus development and showed that PLC is the effector of 5-HT2B during craniofacial development. Our results unveiled an unsuspected role of 5-HT2B in craniofacial development and contribute to our understanding of the interactive network of patterning signals that is involved in the development and evolution of the vertebrate mandibular arch.

MeSH Categories: Animals, Animals, Genetically Modified, Base Sequence, Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism, Facial Bones/embryology/metabolism, Gene Expression Regulation, Developmental, Homeodomain Proteins/genetics/metabolism, In Situ Hybridization, Joints/embryology/metabolism, Neural Crest/cytology/embryology/metabolism, Oligodeoxyribonucleotides, Antisense/genetics, Phospholipase C beta/genetics/metabolism, RNA, Messenger/genetics/metabolism, Receptor, Serotonin, 5-HT2B/antagonists & inhibitors/genetics/*metabolism, Signal Transduction, Skull/embryology/metabolism, Transcription Factors/genetics/metabolism, Xenopus Proteins/genetics/*metabolism, Xenopus laevis/*embryology/genetics/*metabolism

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Mechanosensilla in the adult abdomen of Drosophila: engrailed and slit help to corral the peripheral sensory axons into segmental bundles.

Publication Date: 2010 Sep 1 PMID: 20667917
Authors: Fabre, C. C. - Casal, J. - Lawrence, P. A.
Journal: Development

The abdomen of adult Drosophila bears mechanosensory bristles with axons that connect directly to the CNS, each hemisegment contributing a separate nerve bundle. Here, we alter the amount of Engrailed protein and manipulate the Hedgehog signalling pathway in clones of cells to study their effects on nerve pathfinding within the peripheral nervous system. We find that high levels of Engrailed make the epidermal cells inhospitable to bristle neurons; sensory axons that are too near these cells are either deflected or fail to extend properly or at all. We then searched for the engrailed-dependent agent responsible for these repellent properties. We found slit to be expressed in the P compartment and, using genetic mosaics, present evidence that Slit is the responsible molecule. Blocking the activity of the three Robo genes (putative receptors for Slit) with RNAi supported this hypothesis. We conclude that, during normal development, gradients of Slit protein repel axons away from compartment boundaries - in consequence, the bristles from each segment send their nerves to the CNS in separated sets.

MeSH Categories: Abdomen/physiology, Adaptor Proteins, Signal Transducing/genetics/physiology, Animals, Animals, Genetically Modified, Axons/physiology, Drosophila/genetics/*growth & development/*physiology, Drosophila Proteins/genetics/*physiology, Gene Expression Regulation, Developmental, Genes, Insect, Hedgehog Proteins/genetics/physiology, Homeodomain Proteins/genetics/*physiology, Mechanoreceptors/*physiology, Models, Neurological, Nerve Tissue Proteins/antagonists & inhibitors/genetics/*physiology, Neurogenesis/genetics/*physiology, RNA Interference, Receptors, Cell Surface/genetics/physiology, Receptors, G-Protein-Coupled/genetics/physiology, Receptors, Immunologic/antagonists & inhibitors/genetics/physiology, Transcription Factors/genetics/*physiology

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Transcriptomic landscape of the primitive streak.

Publication Date: 2010 Sep 1 PMID: 20667916
Authors: Alev, C. - Wu, Y. - Kasukawa, T. - Jakt, L. M. - Ueda, H. R. - Sheng, G.
Journal: Development

In birds and mammals, all mesoderm cells are generated from the primitive streak. Nascent mesoderm cells contain unique dorsoventral (D/V) identities according to their relative ingression position along the streak. Molecular mechanisms controlling this initial phase of mesoderm diversification are not well understood. Using the chick model, we generated high-quality transcriptomic datasets of different streak regions and analyzed their molecular heterogeneity. Fifteen percent of expressed genes exhibit differential expression levels, as represented by two major groups (dorsal to ventral and ventral to dorsal). A complete set of transcription factors and many novel genes with strong and region-specific expression were uncovered. Core components of BMP, Wnt and FGF pathways showed little regional difference, whereas their positive and negative regulators exhibited both dorsal-to-ventral and ventral-to-dorsal gradients, suggesting that robust D/V positional information is generated by fine-tuned regulation of key signaling pathways at multiple levels. Overall, our study provides a comprehensive molecular resource for understanding mesoderm diversification in vivo and targeted mesoderm lineage differentiation in vitro.

MeSH Categories: Animals, Avian Proteins/genetics, Body Patterning/genetics, Bone Morphogenetic Proteins/genetics, Chick Embryo, Chromosome Mapping, Fibroblast Growth Factors/genetics, *Gene Expression Profiling, Gene Expression Regulation, Developmental, In Situ Hybridization, Mesoderm/embryology/metabolism, Multigene Family, Oligonucleotide Array Sequence Analysis, Primitive Streak/*embryology/metabolism, Signal Transduction, Transcription Factors/genetics, Transforming Growth Factor beta/genetics, Wnt Proteins/genetics

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The same enhancer regulates the earliest Emx2 expression in caudal forebrain primordium, subsequent expression in dorsal telencephalon and later expression in the cortical ventricular zone.

Publication Date: 2010 Sep 1 PMID: 20667915
Authors: Suda, Y. - Kokura, K. - Kimura, J. - Kajikawa, E. - Inoue, F. - Aizawa, S.
Journal: Development

We have analyzed Emx2 enhancers to determine how Emx2 functions during forebrain development are regulated. The FB (forebrain) enhancer we identified immediately 3' downstream of the last coding exon is well conserved among tetrapods and unexpectedly directed all the Emx2 expression in forebrain: caudal forebrain primordium at E8.5, dorsal telencephalon at E9.5-E10.5 and the cortical ventricular zone after E12.5. Otx, Tcf, Smad and two unknown transcription factor binding sites were essential to all these activities. The mutant that lacked this enhancer demonstrated that Emx2 expression under the enhancer is solely responsible for diencephalon development. However, in telencephalon, the FB enhancer did not have activities in cortical hem or Cajal-Retzius cells, nor was its activity in the cortex graded. Emx2 expression was greatly reduced, but persisted in the telencephalon of the enhancer mutant, indicating that there exists another enhancer for Emx2 expression unique to mammalian telencephalon.

MeSH Categories: Animals, Base Sequence, Binding Sites/genetics, Cerebral Cortex/embryology/metabolism, Conserved Sequence, DNA/genetics/metabolism, DNA Primers/genetics, *Enhancer Elements, Genetic, Female, Gene Expression Regulation, Developmental, Gestational Age, Homeodomain Proteins/*genetics, Mice, Mice, Knockout, Mice, Mutant Strains, Mice, Transgenic, Molecular Sequence Data, Mutation, Otx Transcription Factors/metabolism, Phenotype, Pregnancy, Prosencephalon/*embryology/*metabolism, Sequence Homology, Nucleic Acid, Smad Proteins/metabolism, Species Specificity, TCF Transcription Factors/metabolism, Telencephalon/embryology/metabolism, Transcription Factors/deficiency/*genetics, Xenopus/genetics

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Hfp inhibits Drosophila myc transcription and cell growth in a TFIIH/Hay-dependent manner.

Publication Date: 2010 Sep 1 PMID: 20667914
Authors: Mitchell, N. C. - Johanson, T. M. - Cranna, N. J. - Er, A. L. - Richardson, H. E. - Hannan, R. D. - Quinn, L. M.
Journal: Development

An unresolved question regarding the RNA-recognition motif (RRM) protein Half pint (Hfp) has been whether its tumour suppressor behaviour occurs by a transcriptional mechanism or via effects on splicing. The data presented here demonstrate that Hfp achieves cell cycle inhibition via an essential role in the repression of Drosophila myc (dmyc) transcription. We demonstrate that regulation of dmyc requires interaction between the transcriptional repressor Hfp and the DNA helicase subunit of TFIIH, Haywire (Hay). In vivo studies show that Hfp binds to the dmyc promoter and that repression of dmyc transcription requires Hfp. In addition, loss of Hfp results in enhanced cell growth, which depends on the presence of dMyc. This is consistent with Hfp being essential for inhibition of dmyc transcription and cell growth. Further support for Hfp controlling dmyc transcriptionally comes from the demonstration that Hfp physically and genetically interacts with the XPB helicase component of the TFIIH transcription factor complex, Hay, which is required for normal levels of dmyc expression, cell growth and cell cycle progression. Together, these data demonstrate that Hfp is crucial for repression of dmyc, suggesting that a transcriptional, rather than splicing, mechanism underlies the regulation of dMyc and the tumour suppressor behaviour of Hfp.

MeSH Categories: 5' Untranslated Regions, Animals, Animals, Genetically Modified, Base Sequence, Cell Proliferation, DNA Helicases/metabolism, DNA Primers/genetics, DNA-Binding Proteins/*genetics/*metabolism, Drosophila/*cytology/genetics/growth & development/*metabolism, Drosophila Proteins/antagonists & inhibitors/*genetics/*metabolism, *Genes, Insect, *Genes, myc, Guanine Nucleotide Exchange Factors/antagonists &, inhibitors/genetics/*metabolism, Models, Biological, Promoter Regions, Genetic, RNA Interference, S Phase, Signal Transduction, Transcription Factor TFIIH/*metabolism, Transcription Factors/*genetics, Transcription, Genetic, Wing/growth & development/metabolism

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Pph13 and orthodenticle define a dual regulatory pathway for photoreceptor cell morphogenesis and function.

Publication Date: 2010 Sep 1 PMID: 20667913
Authors: Mishra, M. - Oke, A. - Lebel, C. - McDonald, E. C. - Plummer, Z. - Cook, T. A. - Zelhof, A. C.
Journal: Development

The function and integrity of photoreceptor cells are dependent upon the creation and maintenance of specialized apical structures: membrane discs/outer segments in vertebrates and rhabdomeres in insects. We performed a molecular and morphological comparison of Drosophila Pph13 and orthodenticle (otd) mutants to investigate the transcriptional network controlling the late stages of rhabdomeric photoreceptor cell development and function. Although Otd and Pph13 have been implicated in rhabdomere morphogenesis, we demonstrate that it is necessary to remove both factors to completely eliminate rhabdomere formation. Rhabdomere absence is not the result of degeneration or a failure of initiation, but rather the inability of the apical membrane to transform and elaborate into a rhabdomere. Transcriptional profiling revealed that Pph13 plays an integral role in promoting rhabdomeric photoreceptor cell function. Pph13 regulates Rh2 and Rh6, and other phototransduction genes, demonstrating that Pph13 and Otd control a distinct subset of Rhodopsin-encoding genes in adult visual systems. Bioinformatic, DNA binding and transcriptional reporter assays showed that Pph13 can bind and activate transcription via a perfect Pax6 homeodomain palindromic binding site and the Rhodopsin core sequence I (RCSI) found upstream of Drosophila Rhodopsin genes. In vivo studies indicate that Pph13 is necessary and sufficient to mediate the expression of a multimerized RCSI reporter, a marker of photoreceptor cell specificity previously suggested to be regulated by Pax6. Our studies define a key transcriptional regulatory pathway that is necessary for late Drosophila photoreceptor development and will serve as a basis for better understanding rhabdomeric photoreceptor cell development and function.

MeSH Categories: Animals, Animals, Genetically Modified, Base Sequence, Binding Sites/genetics, DNA/genetics/metabolism, Drosophila Proteins/genetics/*physiology, Drosophila melanogaster/cytology/genetics/*growth &, development/*physiology, Gene Expression Profiling, Gene Expression Regulation, Developmental, Genes, Insect, Homeodomain Proteins/genetics/*physiology, Microscopy, Electron, Transmission, Mutation, Photoreceptor Cells, Invertebrate/*cytology/*physiology/ultrastructure, Promoter Regions, Genetic, Rhodopsin/genetics/physiology

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Developmental RNA processing of 3'UTRs in Hox mRNAs as a context-dependent mechanism modulating visibility to microRNAs.

Publication Date: 2010 Sep 1 PMID: 20667912
Authors: Thomsen, S. - Azzam, G. - Kaschula, R. - Williams, L. S. - Alonso, C. R.
Journal: Development

The Drosophila Hox gene Ultrabithorax (Ubx) controls the development of thoracic and abdominal segments, allocating segment-specific features to different cell lineages. Recent studies have shown that Ubx expression is post-transcriptionally regulated by two microRNAs (miRNAs), miR-iab4 and miR-iab8, acting on target sites located in the 3' untranslated regions (UTRs) of Ubx mRNAs. Here, we show that during embryonic development Ubx produces mRNAs with variable 3'UTRs in different regions of the embryo. Analysis of the resulting remodelled 3'UTRs shows that each species harbours different sets of miRNA target sites, converting each class of Ubx mRNA into a considerably different substrate for miRNA regulation. Furthermore, we show that the distinct developmental distributions of Ubx 3'UTRs are established by a mechanism that is independent of miRNA regulation and therefore are not the consequence of miR-iab4/8-mediated RNA degradation acting on those sensitive mRNA species; instead, we propose that this is a hard-wired 3'UTR processing system that is able to regulate target mRNA visibility to miRNAs according to developmental context. We show that reporter constructs that include Ubx short and long 3'UTR sequences display differential expression within the embryonic central nervous system, and also demonstrate that mRNAs of three other Hox genes suffer similar and synchronous developmental 3'UTR processing events during embryogenesis. Our work thus reveals that developmental RNA processing of 3'UTR sequences is a general molecular strategy used by a key family of developmental regulators so that their transcripts can display different levels of visibility to miRNA regulation according to developmental cues.

MeSH Categories: 3' Untranslated Regions, Animals, Animals, Genetically Modified, Drosophila/*embryology/genetics/*metabolism, Drosophila Proteins/*genetics, Gene Expression Regulation, Developmental, Genes, Insect, Homeodomain Proteins/*genetics, MicroRNAs/genetics/*metabolism, Models, Biological, RNA Processing, Post-Transcriptional, RNA, Messenger/genetics/*metabolism, Transcription Factors/*genetics

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H3K27me3 regulates BMP activity in developing spinal cord.

Publication Date: 2010 Sep 1 PMID: 20667911
Authors: Akizu, N. - Estaras, C. - Guerrero, L. - Marti, E. - Martinez-Balbas, M. A.
Journal: Development

During spinal cord development, the combination of secreted signaling proteins and transcription factors provides information for each neural type differentiation. Studies using embryonic stem cells show that trimethylation of lysine 27 of histone H3 (H3K27me3) contributes to repression of many genes key for neural development. However, it remains unclear how H3K27me3-mediated mechanisms control neurogenesis in developing spinal cord. Here, we demonstrate that H3K27me3 controls dorsal interneuron generation by regulation of BMP activity. Our study indicates that expression of Noggin, a BMP extracellular inhibitor, is repressed by H3K27me3. Moreover, we show that Noggin expression is induced by BMP pathway signaling, generating a negative-feedback regulatory loop. In response to BMP pathway activation, JMJD3 histone demethylase interacts with the Smad1/Smad4 complex to demethylate and activate the Noggin promoter. Together, our data reveal how the BMP signaling pathway restricts its own activity in developing spinal cord by modulating H3K27me3 levels at the Noggin promoter.

MeSH Categories: Animals, Animals, Genetically Modified, Base Sequence, Bone Morphogenetic Proteins/*metabolism, Carrier Proteins/genetics/metabolism, Cell Differentiation, Cell Proliferation, Chick Embryo, DNA Primers/genetics, Epigenesis, Genetic, Histones/chemistry/*metabolism, Humans, Methylation, Models, Neurological, Neurogenesis, Promoter Regions, Genetic, RNA, Messenger/genetics/metabolism, Signal Transduction, Smad Proteins/genetics/metabolism, Spinal Cord/cytology/*embryology/*metabolism

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Strigolactones enhance competition between shoot branches by dampening auxin transport.

Publication Date: 2010 Sep 1 PMID: 20667910
Authors: Crawford, S. - Shinohara, N. - Sieberer, T. - Williamson, L. - George, G. - Hepworth, J. - Muller, D. - Domagalska, M. A. - Leyser, O.
Journal: Development

Strigolactones (SLs), or their derivatives, were recently demonstrated to act as endogenous shoot branching inhibitors, but their biosynthesis and mechanism of action are poorly understood. Here we show that the branching phenotype of mutants in the Arabidopsis P450 family member, MAX1, can be fully rescued by strigolactone addition, suggesting that MAX1 acts in SL synthesis. We demonstrate that SLs modulate polar auxin transport to control branching and that both the synthetic SL GR24 and endogenous SL synthesis significantly reduce the basipetal transport of a second branch-regulating hormone, auxin. Importantly, GR24 inhibits branching only in the presence of auxin in the main stem, and enhances competition between two branches on a common stem. Together, these results support two current hypotheses: that auxin moving down the main stem inhibits branch activity by preventing the establishment of auxin transport out of axillary branches; and that SLs act by dampening auxin transport, thus enhancing competition between branches.

MeSH Categories: Arabidopsis/genetics/*growth & development/*metabolism, Arabidopsis Proteins/genetics/metabolism, Base Sequence, Biological Transport, Active/drug effects, DNA Primers/genetics, Genes, Plant, Indoleacetic Acids/*metabolism, Lactones/*metabolism/pharmacology, Membrane Transport Proteins/genetics/metabolism, Mutation, Phenotype, Plant Growth Regulators/*metabolism, Plant Shoots/drug effects/*growth & development/*metabolism, Plants, Genetically Modified, Xylem/drug effects/metabolism

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Temporal specification of blood progenitors from mouse embryonic stem cells and induced pluripotent stem cells.

Publication Date: 2010 Sep 1 PMID: 20659975
Authors: Irion, S. - Clarke, R. L. - Luche, H. - Kim, I. - Morrison, S. J. - Fehling, H. J. - Keller, G. M.
Journal: Development

The efficient and reproducible generation of differentiated progenitors from pluripotent stem cells requires the recapitulation of appropriate developmental stages and pathways. Here, we have used the combination of activin A, BMP4 and VEGF under serum-free conditions to induce hematopoietic differentiation from both embryonic and induced pluripotent stem cells, with the aim of modeling the primary sites of embryonic hematopoiesis. We identified two distinct Flk1-positive hematopoietic populations that can be isolated based on temporal patterns of emergence. The earliest arising population displays characteristics of yolk sac hematopoiesis, whereas a late developing Flk1-positive population appears to reflect the para-aortic splanchnopleura hematopoietic program, as it has reduced primitive erythroid capacity and substantially enhanced myeloid and lymphoid potential compared with the earlier wave. These differences between the two populations are accompanied by differences in the expression of Sox17 and Hoxb4, as well as in the cell surface markers AA4.1 and CD41. Together, these findings support the interpretation that the two populations are representative of the early sites of mammalian hematopoiesis.

MeSH Categories: Activins/administration & dosage, Animals, Bone Morphogenetic Protein 4/administration & dosage, Cell Differentiation/drug effects/physiology, Cell Line, Embryonic Stem Cells/*cytology/drug effects/metabolism, Gene Expression, HMGB Proteins/genetics/metabolism, Hematopoiesis/drug effects/genetics/*physiology, Hematopoietic Stem Cells/*cytology/drug effects/metabolism, Homeodomain Proteins/genetics/metabolism, Lymphopoiesis/drug effects/genetics/physiology, Membrane Glycoproteins/metabolism, Mice, Models, Biological, Platelet Membrane Glycoprotein IIb/metabolism, Pluripotent Stem Cells/*cytology/drug effects/metabolism, Receptors, Complement/metabolism, Recombinant Proteins/administration & dosage, SOXF Transcription Factors/genetics/metabolism, Time Factors, Transcription Factors/genetics/metabolism, Vascular Endothelial Growth Factor A/administration & dosage, Vascular Endothelial Growth Factor Receptor-2/metabolism

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Arginine methylation of SmB is required for Drosophila germ cell development.

Publication Date: 2010 Sep 1 PMID: 20659974
Authors: Anne, J.
Journal: Development

Sm proteins constitute the common core of spliceosomal small nuclear ribonucleoproteins. Although Sm proteins are known to be methylated at specific arginine residues within the C-terminal arginine-glycine dipeptide (RG) repeats, the biological relevance of these modifications remains unknown. In this study, a tissue-specific function of arginine methylation of the SmB protein was identified in Drosophila. Analysis of the distribution of SmB during oogenesis revealed that this protein accumulates at the posterior pole of the oocyte, a cytoplasmic region containing the polar granules, which are necessary for the formation of primordial germ cells. The pole plasm localisation of SmB requires the methylation of arginine residues in its RG repeats by the Capsuleen-Valois methylosome complex. Functional studies showed that the methylation of these arginine residues is essential for distinct processes of the germline life cycle, including germ cell formation, migration and differentiation. In particular, the methylation of a subset of these arginine residues appears essential for the anchoring of the polar granules at the posterior cortex of the oocyte, whereas the methylation of another subset controls germ cell migration during embryogenesis. These results demonstrate a crucial role of arginine methylation in directing the subcellular localisation of SmB and that this modification contributes specifically to the establishment and development of germ cells.

MeSH Categories: Animals, Animals, Genetically Modified, Arginine/metabolism, Cell Polarity, Drosophila/*embryology/genetics/*metabolism, Drosophila Proteins/chemistry/genetics/*metabolism, Green Fluorescent Proteins/genetics/metabolism, Methylation, Models, Biological, Mutagenesis, Site-Directed, Oocytes/metabolism, Oogenesis/genetics/*physiology, Recombinant Fusion Proteins/chemistry/genetics/metabolism, Ribonucleoproteins, Small Nuclear/chemistry/genetics/*metabolism, Subcellular Fractions/metabolism

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Functional full-term placentas formed from parthenogenetic embryos using serial nuclear transfer.

Publication Date: 2010 Sep 1 PMID: 20659973
Authors: Hikichi, T. - Ohta, H. - Wakayama, S. - Wakayama, T.
Journal: Development

Mammalian parthenogenetic embryos invariably die in mid-gestation from imprinted gene defects and placental hypoplasia. Based on chimera experiments, trophoblastic proliferation is supposed to be inhibited in the absence of a male genome. Here, we show that parthenogenetic mouse embryonic cell nuclei can be reprogrammed by serial rounds of nuclear transfer without using any genetic modification. The durations of survival in uteri of cloned foetuses derived from green fluorescent protein (GFP)-labelled parthenogenetic cell nuclei were extended with repeated nuclear transfers. After five repeats, live cloned foetuses were obtained up to day 14.5 of gestation; however, they did not survive longer even when we repeated nuclear transfer up to nine times. All foetuses showed intestinal herniation and possessed well-expanded large placentas. When embryonic stem (ES) cells derived from fertilised embryos were aggregated with the cloned embryos, full-term offspring with large placentas were obtained from the chimeric embryos. Those placentas were derived from parthenogenetic cell nuclei, judging from GFP expression. The patterns of imprinted gene expression and methylation status were similar to their parthenogenetic origin, except for Peg10, which showed the same level as in the normal placenta. These results suggest that there is a limitation for foetal development in the ability to reprogramme imprinted genes by repeated rounds of nuclear transfer. However, the placentas of parthenogenetic embryos can escape epigenetic regulation when developed using nuclear transfer techniques and can support foetal development to full gestation.

MeSH Categories: Animals, Base Sequence, Cell Differentiation, Cell Line, Cloning, Organism, DNA Methylation, DNA Primers/genetics, Embryonic Development, Embryonic Stem Cells/cytology, Female, Gene Expression, Genomic Imprinting, Male, Mice, Mice, Inbred C57BL, Mice, Inbred DBA, Mice, Inbred ICR, Mice, Transgenic, Nuclear Reprogramming/genetics/physiology, *Nuclear Transfer Techniques, Parthenogenesis/genetics/*physiology, Placenta/cytology/*growth & development/physiology, Pluripotent Stem Cells/cytology, Pregnancy, Totipotent Stem Cells/cytology, Transplantation Chimera

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