bluuebunny/crossref_metadata_2025 huggingface dataset:
Overexpression of Bcl-w in the Testis Disrupts Spermatogenesis: Revelation of a Role of BCL-W in Male Germ Cell Cycle Control
Authors: Yan Wei , Huang Jun-Xing , Lax Anna-Stina , Pelliniemi Lauri , Salminen Eeva , Poutanen Matti , Toppari Jorma
Published: June 2003

To explore physiological roles of BCL-W, a prosurvival member of the BCL-2 protein family, we generated transgenic (TG) mice overexpressing Bcl-w driven by a chicken β-actin promoter. Male Bcl-w TG mice developed normally but were infertile. The adult TG testes displayed disrupted spermatogenesis with various severities ranging from thin seminiferous epithelium containing less germ cells to Sertoli cell-only appearance. No overpopulation of any type of germ cells was observed during testicular development. In contrast, the developing TG testes displayed decreased number of spermatogonia, degeneration, and detachment of spermatocytes and Sertoli cell vacuolization. The proliferative activity of germ cells was significantly reduced during testicular development and spermatogenesis, as determined by in vivo and in vitro 5′-bromo-2′deoxyuridine incorporation assays. Sertoli cells were structurally and functionally normal. The degenerating germ cells were TUNEL-negative and no typical apoptotic DNA ladder was detected. Our data suggest that regulated spatial and temporal expression of BCL-W is required for normal testicular development and spermatogenesis, and overexpression of BCL-W inhibits germ cell cycle entry and/or cell cycle progression leading to disrupted spermatogenesis.
Review of the in Vivo Functions of the p160 Steroid Receptor Coactivator Family
Authors: Xu Jianming , Li Qingtian
Published: June 2003

The p160 steroid receptor coactivator (SRC) gene family contains three homologous members, which serve as transcriptional coactivators for nuclear receptors and certain other transcription factors. These coactivators interact with ligand-bound nuclear receptors to recruit histone acetyltransferases and methyltransferases to specific enhancer/promotor regions, which facilitates chromatin remodeling, assembly of general transcription factors, and transcription of target genes. This minireview summarizes our current knowledge about the molecular structures, molecular mechanisms, temporal and spatial expression patterns, and biological functions of the SRC family. In particular, this article highlights the roles of SRC-1 (NCoA-1), SRC-2 (GRIP1, TIF2, or NCoA-2) and SRC-3 (p/CIP, RAC3, ACTR, AIB1, or TRAM-1) in development, organ function, endocrine regulation, and nuclear receptor function, which are defined by characterization of the genetically manipulated animal models. Furthermore, this article also reviews our current understanding of the role of SRC-3 in breast cancer and discusses possible mechanisms for functional specificity and redundancy among SRC family members.
Formation of the adult pigment pattern in zebrafish requiresleopardandobelixdependent cell interactions
Authors: Maderspacher Florian , Nüsslein-Volhard Christiane
Published: June 2003

Colour patterns are a prominent feature of many animals and are of high evolutionary relevance. In zebrafish, the adult pigment pattern comprises alternating stripes of two pigment cell types, melanophores and xanthophores. How the stripes are defined and a straight boundary is formed remains elusive. We find that mutants lacking one pigment cell type lack a striped pattern. Instead, cells of one type form characteristic patterns by homotypic interactions. Using mosaic analysis, we show that juxtaposition of melanophores and xanthophores suffices to restore stripe formation locally. Based on this, we have analysed the pigment pattern of two adult specific mutants: leopard and obelix. We demonstrate that obelix is required in melanophores to promote their aggregation and controls boundary integrity. By contrast, leopard regulates homotypic interaction within both melanophores and xanthophores, and interaction between the two, thus controlling boundary shape. These findings support a view in which cell-cell interactions among pigment cells are the major driving force for adult pigment pattern formation.
The zebrafishfgf24mutant identifies an additional level of Fgf signaling involved in vertebrate forelimb initiation
Authors: Fischer Sabine , Draper Bruce W. , Neumann Carl J.
Published: June 2003

The development of vertebrate limb buds is triggered in the lateral plate mesoderm by a cascade of genes, including members of the Fgf and Wnt families,as well as the transcription factor tbx5. Fgf8, which is expressed in the intermediate mesoderm, is thought to initiate forelimb formation by activating wnt2b, which then induces the expression of tbx5in the adjacent lateral plate mesoderm. Tbx5, in turn, is required for the activation of fgf10, which relays the limb inducing signal to the overlying ectoderm. We show that the zebrafish fgf24 gene, which belongs to the Fgf8/17/18 subfamily of Fgf ligands, acts downstream of tbx5 to activate fgf10 expression in the lateral plate mesoderm. We also show that fgf24 activity is necessary for the migration of tbx5-expressing cells to the fin bud, and for the activation of shh, but not hand2, expression in the posterior fin bud.
Wnt signalling regulates myogenic differentiation in the developing avian wing
Authors: Anakwe Kelly , Robson Lesley , Hadley Julia , Buxton Paul , Church Vicki , Allen Steve , Hartmann Christine , Harfe Brian , Nohno Tsutomu , Brown Anthony M. C. , Evans Darrell J. R. , Francis-West Philippa
Published: June 2003

The limb musculature arises by delamination of premyogenic cells from the lateral dermomyotome. Initially the cells express Pax3 but, upon entering the limb bud, they switch on the expression of MyoD and Myf5 and undergo terminal differentiation into slow or fast fibres,which have distinct contractile properties that determine how a muscle will function. In the chick, the premyogenic cells express the Wnt antagonist Sfrp2, which is downregulated as the cells differentiate, suggesting that Wnts might regulate myogenic differentiation. Here, we have investigated the role of Wnt signalling during myogenic differentiation in the developing chick wing bud by gain- and loss-of-function studies in vitro and in vivo. We show that Wnt signalling changes the number of fast and/or slow fibres. For example, in vivo, Wnt11 decreases and increases the number of slow and fast fibres,respectively, whereas overexpression of Wnt5a or a dominant-negative Wnt11 protein have the opposite effect. The latter shows that endogenous Wnt11 signalling determines the number of fast and slow myocytes. The distinct effects of Wnt5a and Wnt11 are consistent with their different expression patterns, which correlate with the ultimate distribution of slow and fast fibres in the wing. Overexpression of activated calmodulin kinase II mimics the effect of Wnt5a, suggesting that it uses this pathway. Finally, we show that overexpression of the Wnt antagonist Sfrp2 and ΔLef1 reduces the number of myocytes. In Sfrp2-infected limbs, the number of Pax3 expressing cells was increased, suggesting that Sfrp2 blocks myogenic differentiation. Therefore, Wnt signalling modulates both the number of terminally differentiated myogenic cells and the intricate slow/fast patterning of the limb musculature.
Regulation of myosin expression during myotome formation
Authors: Sacks Loren D. , Cann Gordon M. , Nikovits William , Conlon Sandra , Espinoza Nora R. , Stockdale Frank E.
Published: June 2003

The first skeletal muscle fibers to form in vertebrate embryos appear in the somitic myotome. PCR analysis and in situ hybridization with isoform-specific probes reveal differences in the temporal appearance and spatial distribution of fast and slow myosin heavy chainmRNA transcripts within myotomal fibers. Embryonic fast myosin heavy chain was the first isoform expressed, followed rapidly by slow myosin heavy chains 1 and 3, with slow myosin heavy chain 2 appearing several hours later. Neonatal fast myosin heavy chain is not expressed in myotomal fibers. Although transcripts of embryonic fast myosin heavy chain were always distributed throughout the length of myotomal fibers, the mRNA for each slow myosin heavy chain isoform was initially restricted to the centrally located myotomal fiber nuclei. As development proceeded, slow myosin heavy chain transcripts spread throughout the length of myotomal fibers in order of their appearance. Explants of segments from embryos containing neural tube, notochord and somites 7-10, when incubated overnight, become innervated by motor neurons from the neural tube and express all four myosin heavy chain genes. Removal of the neural tube and/or notochord from explants prior to incubation or addition of d-tubocurare to intact explants prevented expression of slow myosin chain 2 but expression of genes encoding the other myosin heavy chain isoforms was unaffected. Thus, expression of slow myosin heavy chain 2 is dependent on functional innervation, whereas expression of embryonic fast and slow myosin heavy chain 1 and 3are innervation independent. Implantation of sonic-hedgehog-soaked beads in vivo increased the accumulation of both fast and slow myosin heavy chain transcripts, as well as overall myotome size and individual fiber size, but had no effect on myotomal fiber phenotype. Transcripts encoding embryonic fast myosin heavy chain first appear ventrolaterally in the myotome, whereas slow myosin heavy chain transcripts first appear in fibers positioned midway between the ventrolateral and dorsomedial lips of the myotome. Therefore, models of epaxial myotome formation must account for the positioning of the oldest fibers in the more ventral-lateral region of the myotome and the youngest fibers in the dorsomedial region.
TheCaenorhabditis elegansems class homeobox gene ceh-2 is required for M3 pharynx motoneuron function
Authors: Aspöck Gudrun , Ruvkun Gary , Bürglin Thomas R.
Published: June 2003

Several homeobox genes, for example those of the ems class, play important roles in animal head development. We report on the expression pattern and function of ceh-2, the Caenorhabditis elegans ems/Emx ortholog. CEH-2 protein is restricted to the nuclei of one type of small muscle cell, one type of epithelial cell, and three types of neurons in the anterior pharynx in the head. We have generated a deletion allele of ceh-2 that removes the homeobox. Animals homozygous for this deletion are viable and fertile, but grow slightly slower and lay fewer eggs than wild type. We assayed the function of two types of pharynx neurons that express ceh-2, the pairs M3 and NSM. M3 activity is substantially reduced in electropharyngeograms of ceh-2 deletion mutants; this defect can account for the observed retardation in larval development, as M3 activity is known to be necessary for effective feeding. NSM function and metabolism are normal based on the assays used. All cells that express ceh-2 in wild type are present in the ceh-2mutant and have normal morphologies. Therefore, unlike other ems/Emxgenes, ceh-2 seems to be important for a late differentiation step and not for neuron specification or regional patterning. Because the CEH-2 homeodomain is well conserved, we tested whether ceh-2 can rescue ems- brain defects in Drosophila, despite the apparent differences in biological roles. We found that the C. elegans ems ortholog is able to substitute for fly ems in brain development, indicating that sequence conservation rather than conservation of biological function is important.
Analysis of a key regulatory region upstream of theMyf5gene reveals multiple phases of myogenesis, orchestrated at each site by a combination of elements dispersed throughout the locus
Authors: Hadchouel Juliette , Carvajal Jaime J. , Daubas Philippe , Bajard Lola , Chang Ted , Rocancourt Didier , Cox David , Summerbell Dennis , Tajbakhsh Shahragim , Rigby Peter W. J. , Buckingham Margaret
Published: June 2003

Myf5 is the first myogenic regulatory factor to be expressed in the mouse embryo and it determines the entry of cells into the skeletal muscle programme. A region situated between -58 kb and -48 kb from the gene directs Myf5 transcription at sites where muscles will form. We now show that this region consists of a number of distinct regulatory elements that specifically target sites of myogenesis in the somite, limbs and hypoglossal cord, and also sites of Myf5 transcription in the central nervous system. Deletion of these sequences in the context of the locus shows that elements within the region are essential, and also reveals the combinatorial complexity of the transcriptional regulation of Myf5. Both within the-58 kb to -48 kb region and elsewhere in the locus, multiple sequences are present that direct transcription in subdomains of a single site during development, thus revealing distinct phases of myogenesis when subpopulations of progenitor cells enter the programme of skeletal muscle differentiation.
Rb and p107 are required for normal cerebellar development and granule cell survival but not for Purkinje cell persistence
Authors: Marino Silvia , Hoogervoorst Dennis , Brandner Sebastian , Berns Anton
Published: June 2003

The involvement of the retinoblastoma gene product (Rb) and its family members (p107 and p130) in cell cycle exit and terminal differentiation of neural precursor cells has been demonstrated in vitro. To investigate the roles of Rb and p107 in growth, differentiation and apoptosis in the developing and mature cerebellum, we selectively inactivated either Rb alone or in combination with p107 in cerebellar precursor cells or in Purkinje cells. In our mouse models, we show that (1) Rb is required for differentiation, cell cycle exit and survival of granule cell precursors; (2)p107 can not fully compensate for the loss of Rb function in granule cells;(3) Rb and p107 are not required for differentiation and survival of Purkinje cells during embryonic and early postnatal development; (4) Rb function in Purkinje cells is cell autonomous; and (5) loss of Rb deficient CNS precursor cells is mediated by p53-independent apoptosis.
Mesenchymal patterning byHoxa2requires blocking Fgf-dependent activation ofPtx1
Authors: Bobola Nicoletta , Carapuço Marta , Ohnemus Sabine , Kanzler Benoît , Leibbrandt Andreas , Neubüser Annette , Drouin Jacques , Mallo Moisés
Published: June 2003

Hox genes are known key regulators of embryonic segmental identity, but little is known about the mechanisms of their action. To address this issue,we have analyzed how Hoxa2 specifies segmental identity in the second branchial arch. Using a subtraction approach, we found that Ptx1 was upregulated in the second arch mesenchyme of Hoxa2 mutants. This upregulation has functional significance because, in Hoxa2-/-;Ptx1-/- embryos, the Hoxa2-/- phenotype is partially reversed. Hoxa2interferes with the Ptx1 activating process, which is dependent on Fgf signals from the epithelium. Consistently, Lhx6, another target of Fgf8 signaling, is also upregulated in the Hoxa2-/-second arch mesenchyme. Our findings have important implications for the understanding of developmental processes in the branchial area and suggest a novel mechanism for mesenchymal patterning by Hox genes that acts to define the competence of mesenchymal cells to respond to skeletogenic signals.
Fgf3andFgf10are required for mouse otic placode induction
Authors: Wright Tracy J. , Mansour Suzanne L.
Published: June 2003

The inner ear, which contains the sensory organs specialised for audition and balance, develops from an ectodermal placode adjacent to the developing hindbrain. Tissue grafting and recombination experiments suggest that placodal development is directed by signals arising from the underlying mesoderm and adjacent neurectoderm. In mice, Fgf3 is expressed in the neurectoderm prior to and concomitant with placode induction and otic vesicle formation,but its absence affects only the later stages of otic vesicle morphogenesis. We show here that mouse Fgf10 is expressed in the mesenchyme underlying the prospective otic placode. Embryos lacking both Fgf3and Fgf10 fail to form otic vesicles and have aberrant patterns of otic marker gene expression, suggesting that FGF signals are required for otic placode induction and that these signals emanate from both the hindbrain and mesenchyme. These signals are likely to act directly on the ectoderm, as double mutant embryos showed normal patterns of gene expression in the hindbrain. Cell proliferation and survival were not markedly affected in double mutant embryos, suggesting that the major role of FGF signals in otic induction is to establish normal patterns of gene expression in the prospective placode. Finally, examination of embryos carrying three out of the four mutant Fgf alleles revealed intermediate phenotypes, suggesting a quantitative requirement for FGF signalling in otic vesicle formation.
Acceleration of human myoblast fusion by depolarization: graded Ca2+ signals involved
Authors: Liu Jian-Hui , König Stéphane , Michel Marlène , Arnaudeau Serge , Fischer-Lougheed Jacqueline , Bader Charles R. , Bernheim Laurent
Published: June 2003

We have previously shown that human myoblasts do not fuse when their voltage fails to reach the domain of a window T-type Ca2+ current. We demonstrate, by changing the voltage in the window domain, that the Ca2+ signal initiating fusion is not of the all-or-none type, but can be graded and is interpreted as such by the differentiation program. This was carried out by exploiting the properties of human ether-à-go-go related gene K+ channels that we found to be expressed in human myoblasts. Methanesulfonanilide class III antiarrhythmic agents or antisense-RNA vectors were used to suppress completely ether-à-go-go related gene current. Both procedures induced a reproducible depolarization from -74 to -64 mV, precisely in the window domain where the T-type Ca2+ current increases with voltage. This 10 mV depolarization raised the cytoplasmic free Ca2+ concentration, and triggered a tenfold acceleration of myoblast fusion. Our results suggest that any mechanism able to modulate intracellular Ca2+ concentration could affect the rate of myoblast fusion.
Establishment of POP-1 asymmetry in earlyC. elegansembryos
Authors: Park Frederick D. , Priess James R.
Published: June 2003

In Caenhorabtidis elegans embryos, the nuclei of sister cells that are born from anterior/posterior divisions show an invariant high/low asymmetry, respectively, in their level of the transcription factor POP-1. Previous studies have shown that POP-1 asymmetry between the daughters of an embryonic cell called EMS results in part from a Wnt-like signal provided by a neighboring cell, called P2. We identify here additional signaling cells that play a role in POP-1 asymmetry for other early embryonic cells. Some of these cells have signaling properties similar to P2,whereas other cells use apparently distinct signaling pathways. Although cell signaling plays a critical role in POP-1 asymmetry during the first few cell divisions, later embryonic cells have an ability to generate POP-1 asymmetry that appears to be independent of prior Wnt signaling.
Crucial role of TrkB ligands in the survival and phenotypic differentiation of developing locus coeruleus noradrenergic neurons
Authors: Holm Pontus C. , Rodríguez Francisco J. , Kresse Adelheid , Canals Josep M. , Silos-Santiago Inmaculada , Arenas Ernest
Published: June 2003

The role of glial cell-line derived neurotrophic factor (GDNF) and neurotrophins in the development of locus coeruleus noradrenergic neurons was evaluated. We found that two neurotrophic factors previously reported to prevent the degeneration of lesioned adult central noradrenergic neurons, GDNF and neurotrophin 3 (NT3), do not play significant roles in the prenatal development of locus coeruleus noradrenergic neurons, as demonstrated by: (1)the lack of alterations in double Gdnf/Nt3 null mutant mice;and (2) the lack of survival-promoting effects of GDNF and/or NT3 in rat E13.5 primary cultures. In contrast, null mutant mice for TrkB, the tyrosine kinase receptor for brain-derived neurotrophic factor and neurotrophin 4, displayed a clear loss of locus coeruleus noradrenergic neurons. In accordance with this,treatment of rat E13.5 primary cultures with TrkB ligands prevented the early loss of noradrenergic neurons and maintained their survival for up to 6 days in vitro. Moreover, an additional 5-10-fold increase in the number of tyrosine hydroxylase positive noradrenergic neurons was detected after 12 hours in culture. This second effect of TrkB ligands involved neither proliferation nor survival, because the number of BrdU- or TUNEL-positive noradrenergic neurons did not change and the effect was elicited by delayed administration of either factor. Because TrkB ligands increased the number of tyrosine hydroxylase-positive cells expressing Phox2a, a paired homeodomain protein required for the development of locus coeruleus noradrenergic neurons, but did not affect the number of Phox2a-positive tyrosine hydroxylase-negative cells,our results suggest that the second effect of TrkB ligands may involve promoting or inducing a noradrenergic phenotype. In summary, our findings suggest that, unlike NT3 and GDNF, TrkB ligands are required and sufficient to promote the development of central noradrenergic neurons.
TheDrosophiladual-specificity ERK phosphatase DMKP3 cooperates with the ERK tyrosine phosphatase PTP-ER
Authors: Rintelen Felix , Hafen Ernst , Nairz Knud
Published: June 2003

ERK MAP kinase plays a key role in relaying extracellular signals to transcriptional regulation. As different activity levels or the different duration of ERK activity can elicit distinct responses in one and the same cell, ERK has to be under strict positive and negative control. Although numerous genes acting positively in the ERK signaling pathway have been recovered in genetic screens, mutations in genes encoding negative ERK regulators appear underrepresented. We therefore sought to genetically characterize the dual-specificity phosphatase DMKP3. First, we established a novel assay to elucidate the substrate preferences of eukaryotic phosphatases in vivo and thereby confirmed the specificity of DMKP3 as an ERK phosphatase. The Dmkp3 overexpression phenotype characterized in this assay permitted us to isolate Dmkp3 null mutations. By genetic analysis we show that DMKP3 and the tyrosine phosphatase PTP-ER perform partially redundant functions on the same substrate, ERK. DMKP3 functions autonomously in a subset of photoreceptor progenitor cells in eye imaginal discs. In addition, DMKP3 function appears to be required in surrounding non-neuronal cells for ommatidial patterning and photoreceptor differentiation.
Monitoring neural progenitor fate through multiple rounds of division in an intact vertebrate brain
Authors: Lyons David A. , Guy Adam T. , Clarke Jonathan D. W.
Published: June 2003

The behaviour of neural progenitors in the intact vertebrate brain and spinal cord is poorly understood, chiefly because of the inaccessibility and poor optical qualities inherent in many model systems. To overcome these problems we have studied the optically superior brain of the zebrafish embryo and have monitored the in vivo behaviour of fluorescently labelled neural progenitors and their daughter cells throughout a substantial period of hindbrain development. We find the majority (84%) of hindbrain neurons are born from progenitor divisions that generate two neurons and 68% of reconstructed lineage trees contained no asymmetric stem cell-like divisions. No progenitors divided in the manner expected of a classic stem cell; i.e. one that repeatedly self-renews and generates a differentiated cell type by asymmetric division. We also analysed the orientation of progenitor divisions relative to the plane of the ventricular zone (VZ) and find that this does not correlate with the fate of the daughter cells. Our results suggest that in this vertebrate system the molecular determinants that control whether a cell will become a neuron are usually not linked to a mechanism that generates asymmetric divisions.
PVF1, a PDGF/VEGF homolog, is sufficient to guide border cells and interacts genetically with Taiman
Authors: McDonald Jocelyn A. , Pinheiro Elaine M. , Montell Denise J.
Published: June 2003

The border cells of the Drosophila ovary undergo a well-defined and developmentally regulated cell migration. Two signals have previously been shown to control where and when the cells migrate. The steroid hormone ecdysone, acting through its receptor and a coactivator known as Taiman,contributes to regulating the timing of border cell migration. PVF1, a growth factor related to platelet-derived growth factor and vascular-endothelial growth factor, contributes to guiding the border cells to the oocyte. To probe the mechanisms controlling border cell migration further, we performed a screen for genes that exhibit dominant genetic interactions with taiman. We identified 14 genomic regions that interact with taiman. Within one region, we identified Pvf1 as the gene responsible for the interaction. Signaling by PVF1 has been proposed to guide the border cells to their proper target, but ectopic PVF1 has not been tested for its ability to redirect the border cells. We tested the ability of PVF1,as well as other factors such as Gurken, to guide the border cells to new targets. Our results demonstrate that ectopic expression of PVF1 is sufficient to redirect border cells in some egg chambers but that the other factors tested are not. These data suggest that the guidance of border cell migration is robust and that there are likely to be additional factors that contribute to long-range guidance of these cells. In addition, we find that taiman and Pvf1 regulate the dynamic localization of E-cadherin in the border cells, possibly accounting for the interaction between these two pathways.
A dual, non-redundant, role for LIF as a regulator of development and STAT3-mediated cell death in mammary gland
Authors: Kritikou Ekaterini A. , Sharkey Andrew , Abell Kathrine , Came Paul J. , Anderson Elizabeth , Clarkson Richard W. E. , Watson Christine J.
Published: June 2003

STAT3 is the key mediator of apoptosis in mammary gland. We demonstrate here that LIF is the physiological activator of STAT3, because in involuting mammary glands of Lif-;/-; mice, pSTAT3 is absent and the STAT3 target, C/EBPδ, is not upregulated. Similar to Stat3knockouts, Lif-;/-; mammary glands exhibit delayed involution, reduced apoptosis and elevated levels of p53. Significantly, Lif-;/-; glands display precocious development during pregnancy, when pSTAT3 is not normally detected. We show that pERK1/2 is significantly reduced in Lif-;/-; glands at this time,suggesting that at this stage LIF mediates its effects through pERK1/2. Inhibition of LIF-mediated ERK1/2 phosphorylation potentiates the proapoptotic effects of STAT3. LIF therefore signals alternately through ERK1/2, then STAT3, to regulate mammary growth and apoptosis.
Epiblast and primitive-streak origins of the endoderm in the gastrulating chick embryo
Authors: Lawson Aaron , Schoenwolf Gary C.
Published: June 2003

Gastrulation is characterized by the extensive movements of cells. Fate mapping is used to follow such cell movements as they occur over time, and prospective fate maps have been constructed for several stages of the model organisms used in modern studies in developmental biology. In chick embryos,detailed fate maps have been constructed for both prospective mesodermal and ectodermal cells. However, the origin and displacement of the prospective endodermal cells during crucial periods in gastrulation remain unclear. This study had three aims. First, we determined the primitive-streak origin of the endoderm using supravital fluorescent markers, and followed the movement of the prospective endodermal cells as they dispersed to generate the definitive endodermal layer. We show that between stages 3a/b and 4, the intraembryonic definitive endoderm receives contributions mainly from the rostral half of the primitive streak, and that endodermal movements parallel those of ingressing adjacent mesodermal subdivisions. Second, the question of the epiblast origin of the endodermal layer was addressed by precisely labeling epiblast cells in a region known to give rise to prospective somitic cells, and following their movement as they underwent ingression through the primitive streak. We show that the epiblast clearly contributes prospective endodermal cells to the primitive streak, and subsequently to definitive endoderm of the area pellucida. Finally, the relationship between the hypoblast and the definitive endoderm was defined by following labeled rostral primitive-streak cells over a short period of time as they contributed to the definitive endoderm, and combining this with in situ hybridization with a riboprobe for Crescent, a marker of the hypoblast. We show that as the definitive endodermal layer is laid down, there is cell-cell intercalation at its interface with the displaced hypoblast cells. These data were used to construct detailed prospective fate maps of the endoderm in the chick embryo,delineating the origins and migrations of endodermal cells in various rostrocaudal levels of the primitive streak during key periods in early development.
The role of chordin/Bmp signals in mammalian pharyngeal development and DiGeorge syndrome
Authors: Bachiller Daniel , Klingensmith John , Shneyder Natalya , Tran Uyen , Anderson Ryan , Rossant Janet , De Robertis E. M.
Published: June 2003

The chordin/Bmp system provides one of the best examples of extracellular signaling regulation in animal development. We present the phenotype produced by the targeted inactivation of the chordin gene in mouse. Chordin homozygous mutant mice show, at low penetrance, early lethality and a ventralized gastrulation phenotype. The mutant embryos that survive die perinatally,displaying an extensive array of malformations that encompass most features of DiGeorge and Velo-Cardio-Facial syndromes in humans. Chordin secreted by the mesendoderm is required for the correct expression of Tbx1 and other transcription factors involved in the development of the pharyngeal region. The chordin mutation provides a mouse model for head and neck congenital malformations that frequently occur in humans and suggests that chordin/Bmp signaling may participate in their pathogenesis.
Brn3a regulation of TrkA/NGF receptor expression in developing sensory neurons
Authors: Ma Long , Lei Lei , Eng S. Raisa , Turner Eric , Parada Luis F.
Published: June 2003

The TrkA/NGF receptor is essential for the survival and differentiation of sensory neurons. The molecular mechanisms regulating tissue and stage-specific expression of TrkA are largely unknown. The Brn3a POU-domain transcription factor has been implicated in the development of the PNS and proposed as a transcription regulator for TrkA. The molecular mechanisms underlying the regulation of TrkA by Brn3a is unclear. In this study, we provide genetic,transgenic and biochemical evidence that Brn3a binds to novel, specific sites in the 457 bp enhancer that regulates TrkA expression in embryonic sensory neurons. We employ Bax-knockout mice, in which sensory neurons no longer require neurotrophins for survival, to uncouple TrkA-dependent cell death from downregulation of TrkA expression. In addition, when mutagenized, the novel Brn3a-binding sites identified fail to drive appropriate reporter transgene expression in sensory neurons. Thus, TrkA, a gene that is crucial for the differentiation and survival of sensory nociceptive neurons, requires Brn3a to maintain normal transcriptional activity.
Pin1 regulates the timing of mammalian primordial germ cell proliferation
Authors: Atchison Fawn W. , Capel Blanche , Means Anthony R.
Published: June 2003

Primordial germ cells (PGCs) give rise to male and female germ cells to transmit the genome from generation to generation. Defects in PGC development often result in infertility. In the mouse embryo, PGCs undergo proliferation and expansion during and after their migration to the gonads from 8.5 to 13.5 days post coitum (dpc). We show that a peptidyl-prolyl isomerase, Pin1, is involved in the regulation of mammalian PGC proliferation. We discovered that both the male and female Pin1-/- mice had profound fertility defects. Investigation of the reproductive organs revealed significantly fewer germ cells in the adult Pin1-/- testes and ovaries than in wild type or heterozygotes, which resulted from Pin1-/- males and females being born with severely reduced number of gonocytes and oocytes. Further studies in 8.5 to 13.5 dpc Pin1-/- embryos showed that PGCs were allocated properly at the base of the allantois, but their cell expansion was progressively impaired, resulting in a markedly reduced number of PGCs at 13.5 dpc. Analyses using markers of cell cycle parameters and apoptosis revealed that Pin1-/- PGCs did not undergo cell cycle arrest or apoptosis. Instead, Pin1-/- PGCs had a lower BrdU labeling index compared with wild-type PGCs. We conclude that PGCs have a prolonged cell cycle in the absence of Pin1, which translates into fewer cell divisions and strikingly fewer Pin1-/- PGCs by the end of the proliferative phase. These results indicate that Pin1 regulates the timing of PGC proliferation during mouse embryonic development.
Macrophage-mediated corpse engulfment is required for normalDrosophilaCNS morphogenesis
Authors: Sears Heather C. , Kennedy Caleb J. , Garrity Paul A.
Published: June 2003

Cell death plays an essential role in development, and the removal of cell corpses presents an important challenge for the developing organism. Macrophages are largely responsible for the clearance of cell corpses in Drosophila melanogaster and mammalian systems. We have examined the developmental requirement for macrophages in Drosophila and find that macrophage function is essential for central nervous system (CNS)morphogenesis. We generate and analyze mutations in the Pvr locus,which encodes a receptor tyrosine kinase of the PDGF/VEGF family that is required for hemocyte migration. We find that loss of Pvr function causes the mispositioning of glia within the CNS and the disruption of the CNS axon scaffold. We further find that inhibition of hemocyte development or of Croquemort, a receptor required for macrophage-mediated corpse engulfment,causes similar CNS defects. These data indicate that macrophage-mediated clearance of cell corpses is required for proper morphogenesis of the Drosophila CNS.
The role of IFNγ nuclear localization sequence in intracellular function
Authors: Ahmed C. M. Iqbal , Burkhart Marjorie A. , Mujtaba Mustafa G. , Subramaniam Prem S. , Johnson Howard M.
Published: June 2003

Intracellularly expressed interferon γ (IFNγ) has been reported to possess biological activity similar to that of IFNγ added to cells. This study addresses the mechanisms for such similar biological effects. Adenoviral vectors were used to express a non-secreted form of human IFNγ or a non-secreted mutant form in which a previously demonstrated nuclear localization sequence (NLS), 128KTGKRKR134, was replaced with alanines at K and R positions. With the vector expressing non-secreted wild-type IFNγ, biological responses normally associated with extracellular IFNγ, such as antiviral activity and MHC class I upregulation, were observed, although the mutant IFNγ did not possess biological activity. Intracellular human IFNγ possessed biological activity in mouse L cells, which do not recognize extracellularly added human IFNγ. Thus, the biological activity was not due to leakage of IFNγto the surroundings and subsequent interaction with the receptor on the cell surface. Biological function was associated with activation of STAT1αand nuclear translocation of IFNγ, IFNGR1 and STAT1α. Immunoprecipitation of cellular extracts with antibody to the nuclear transporter NPI-1 showed the formation of a complex with IFNγ-IFNGR1-STAT1α. To provide the physiological basis for these effects we show that extracellularly added IFNγ possesses intracellular signaling activity that is NLS dependent, as suggested by our previous studies, and that this activity occurs via the receptor-mediated endocytosis of IFNγ. The data are consistent with previous observations that the NLS of extracellularly added IFNγ plays a role in IFNγ signaling.
Relationship of DNA double-strand breaks to synapsis in Drosophila
Authors: Jang Janet K. , Sherizen Dalia E. , Bhagat Rajal , Manheim Elizabeth A. , McKim Kim S.
Published: June 2003

The relationship between synaptonemal complex formation (synapsis) and double-strand break formation (recombination initiation) differs between organisms. Although double-strand break creation is required for normal synapsis in Saccharomyces cerevisiae and the mouse, it is not necessary for synapsis in Drosophila and Caenorhabditis elegans. To investigate the timing of and requirements for double-strand break formation during Drosophila meiosis, we used an antibody that recognizes a histone modification at double-strand break sites,phosphorylation of HIS2AV (γ-HIS2AV). Our results support the hypothesis that double-strand break formation occurs after synapsis. Interestingly, we detected a low (10-25% of wildtype) number of γ-HIS2AV foci in c(3)G mutants, which fail to assemble synaptonemal complex,suggesting that there may be both synaptonemal complex-dependent and synaptonemal complex-independent mechanisms for generating double-strand breaks. Furthermore, mutations in Drosophila Rad54 (okr) and Rad51 (spnB) homologs cause delayed and prolonged γ-HIS2AV staining, suggesting that double-strand break repair is delayed but not eliminated in these mutants. There may also be an interaction between the recruitment of repair proteins and phosphorylation.
Restricted spatial expression of a high-affinity phosphate transporter in potato roots
Authors: Gordon-Weeks Ruth , Tong Yiping , Davies T. G. Emyr , Leggewie Georg
Published: June 2003

Phosphorus deficiency limits plant growth, and high-affinity phosphate transporters, of the Pht1 family, facilitate phosphate uptake and translocation. The family is subdivided into root specific, phosphate deprivation induced members and those also expressed in leaves. An antibody to StPT2, a potato root specific transporter, detected two bands (52 kDa and 30 kDa) on western blots of root plasma membrane extracts that were most intense in whole extracts from the root tip and slightly increased throughout the root in response to phosphate depletion. RT-PCR, using StPT2 specific primers,confirmed these findings. Low power confocal immunofluorescent images showed StPT2 expression mainly in the elongation zone at the root tip. By contrast, a vacuolar pyrophosphatase and a plasma membrane ATPase antibody labelled the whole root. High power images showed, by comparison with α-tubulin, cell wall and plasma membrane ATPase labelling, that StPT2 was in the epidermal plasma membrane and restricted to the apical surface. This is the first evidence of polar plasma membrane localisation of a plant nutrient transporter and is consistent with a role for StPT2 in phosphate capture and uptake.
Thyroid hormone-upregulated expression of Musashi-1 is specific for progenitor cells of the adult epithelium during amphibian gastrointestinal remodeling
Authors: Ishizuya-Oka Atsuko , Shimizu Katsuhiko , Sakakibara Shin-ichi , Okano Hideyuki , Ueda Shuichi
Published: June 2003

In the amphibian gastrointestine during metamorphosis, the primary (larval)epithelium undergoes apoptosis. By contrast, a small number of undifferentiated cells including stem cells actively proliferate and differentiate into the secondary (adult) epithelium that resembles the mammalian counterpart. In the present study, to clarify whether Musashi-1(Msi-1), an RNA-binding protein, serves as a marker for progenitor cells of the adult epithelium, we chronologically examined Msi-1 expression in the Xenopus laevis gastrointestine by using in situ hybridization and immunohistochemistry. Similar expression profiles of Msi-1 were observed at both mRNA and protein levels. In both the small intestine and the stomach, the transient expression of Msi-1 during metamorphosis spatio-temporally correlated well with active proliferation of the progenitor cells including stem cells of the adult epithelium but did not with apoptosis of the larval epithelium. As the adult progenitor cells differentiated into organ-specific epithelial cells after active proliferation, Msi-1 expression was rapidly downregulated. Therefore, Msi-1 is useful to identify the adult progenitor cells that actively proliferate before final differentiation in the amphibian gastrointestine. Furthermore, our culture experiments have shown that thyroid hormone (TH) organ-autonomously induces Msi-1 expression only in the adult progenitor cells of the X. laevis intestine in vitro as in vivo. However, TH could not induce Msi-1 expression in the intestinal epithelium separated from the connective tissue, where the adult epithelium never developed. These results suggest that Msi-1 expression is upregulated by TH in the adult progenitor cells under the control of the connective tissue and plays important roles in their maintenance and/or active proliferation during amphibian gastrointestinal remodeling.
Intracellular clusterin causes juxtanuclear aggregate formation and mitochondrial alteration
Authors: Debure Laure , Vayssière Jean-Luc , Rincheval Vincent , Loison Fabien , Le Dréan Yves , Michel Denis
Published: June 2003

Clusterin is a puzzling protein upregulated in many diseased tissues,presented as either a survival or a death protein. The role of clusterin might depend on the final maturation and localization of the protein, which can be secreted or reside inside cells, either after in situ synthesis or uptake of extracellular clusterin. We studied the biological effects of intracellular clusterin and observed that clusterin forms containing the α-chain region strongly accumulated in an ubiquitinated form in juxtanuclear aggregates meeting the main criterions of aggresomes and leading to profound alterations of the mitochondrial network. The viability of cells transfected by intracellular forms of clusterin was improved by overexpression of Bcl-2,and caspase inhibition was capable of rescuing cells expressing clusterin,which presented an altered mitochondrial permeability. We propose that,although it might be an inherently pro-survival and anti-apoptotic protein expressed by cells under stress in an attempt to protect themselves, clusterin can become highly cytotoxic when accumulated in the intracellular compartment. This activity might reconcile the opposite purported influences of clusterin on cell survival and explain how clusterin can be causally involved in neurodegeneration.
Differential expression of NO-sensitive guanylyl cyclase subunits during the development of rat cerebellar granule cells: regulation viaN-methyl-D-aspartate receptors
Authors: Jurado Sandra , Sánchez-Prieto José , Torres Magdalena
Published: June 2003

In primary cultures of rat cerebellar granule cells with a functional network of glutamatergic neurons, the expression pattern of the different subunits of nitric-oxide (NO)-sensitive guanylyl cyclase changes during cell differentiation. These cells express the α1,α 2 and β1 subunits of NO-sensitive guanylyl cyclase and synthesize cyclic guanosine monophosphate (cGMP) in response to exogenous or endogenous nitric oxide. In this study, we determined the protein content of the α1 and β1 subunits and quantified α1, α2 and β1mRNA by reverse transcription coupled to a polymerase chain reaction (RT-PCR). Expression of the β1 subunit increased with the degree of cell differentiation, although most marked changes occurred at the α subunit level. In cells freshly isolated from rat pups on postnatal day 7 (P7) the most abundant α subunit was α1, whileα 2 appeared as the predominant subunit of this type in cultured cells. N-methyl-D-aspartate (NMDA) receptor stimulation in 7- or 14-day-cultured cells led to the upregulation of guanylyl cyclase subunit mRNAs; α2 mRNA levels undergoing most significant change. This enhanced subunit expression was accompanied by an increase in the amount of cGMP synthesized in response to NO. Thus, it seems thatα 2 subunits are increasingly expressed as granule cells mature. The presence of this subunit in the guanylyl cyclase heterodimer facilitates its localization at synaptic membranes, where the enzyme acts as a sensor for NO formed by the postsynaptic protein 95 (PSD-95)-associated neuronal NO synthase.
Tyrosine phosphorylation of the CrkII adaptor protein modulates cell migration
Authors: Takino Takahisa , Tamura Masahito , Miyamori Hisashi , Araki Masaru , Matsumoto Kazue , Sato Hiroshi , Yamada Kenneth M.
Published: June 2003

CrkII belongs to a family of adaptor proteins that become tyrosine phosphorylated after various stimuli. We examined the role of CrkII tyrosine phosphorylation in fibronectin-induced cell migration. Overexpression of CrkII inhibited dephosphorylation of focal adhesion components such as p130 Crk-associated substrate (p130cas) and paxillin by protein tyrosine phosphatase 1B (PTP1B). Tyrosine-phosphorylated CrkII was dephosphorylated by PTP1B both in vitro and in vivo, showing for the first time that PTP1B directly dephosphorylates CrkII. A CrkII mutant in which tyrosine residue 221 was substituted by phenylalanine (CrkII-Y221F) could not be tyrosine phosphorylated, and it showed significantly increased binding to p130cas and paxillin. Enhanced binding of CrkII to p130cas has been reported to promote cell migration. Nonphosphorylated CrkII-Y221F promoted HT1080 cell migration on fibronectin,whereas wild-type CrkII did not at moderate expression levels. Moreover,co-expression of CrkII and PTP1B promoted HT1080 cell migration on fibronectin and retained tyrosine phosphorylation and binding of p130cas to CrkII, whereas paxillin tyrosine phosphorylation was reduced. These findings support the concepts that CrkII binding activity is regulated by tyrosine kinases and phosphatases, and that tyrosine phosphorylation of CrkII can downmodulate cell migration mediated by the focal adhesion kinase/p130cas pathway.
Structures and Magnetic Properties of Double Perovskites A2LnMO6 and 6H-Perovskites Ba3LnRu2O9 (A = Sr, Ba; Ln = Y, Lanthanides; M = Nb, Ta, Ru)
Authors: Hinatsu Yukio , Doi Yoshihiro
Published: June 2003

This account describes the synthesis, crystal structures and magnetic properties of double perovskites A2LnMO6 and 6H-perovskites Ba3LnRu2O9 (A = Sr, Ba; Ln = Y, lanthanides; M = Nb, Ta, Ru). The double perovskites A2LnMO6 have two kinds of cations, Ln and M, in the B site of the perovskite ABO3. These cations adopt the alternative ordered arrangement. Measurements of the magnetic susceptibility, specific heat, and powder neutron diffraction showed that all of the A2LnRuO6 compounds exhibited an antiferromagnetic transition at low temperatures and a complex temperature dependence of the magnetic susceptibility below their transition temperatures. In these compounds, the magnetic interaction between the Ln (4f electrons) and Ru (4d electrons) ions via the Ln–O–Ru pathway contributes greatly to their magnetic cooperative phenomena. The structural and magnetic studies for the 6H-perovskites Ba3LnRu2O9 show that the Ln cations occupy the corner-sharing octahedra (LnO6 octahedron) and the Ru cations occupy the face-sharing octahedra (Ru2O9 dimer). The Ba3LnRu2O9 compounds show a characteristic temperature-dependence of the magnetic susceptibilities, that is, a broad maximum at temperatures above 135 K and a magnetic transition at 5–15 K. These magnetic properties mainly reflect two kinds of the magnetic interactions: the interaction between Ln and Ru ions and that between Ru ions in the Ru2O9 dimer.