Targeted Gene Expression in Transgenic Xenopus Using the Binary Gal4-UAS System

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2002  |  Pubmed ID: 11818539

The transgenic technique in Xenopus allows one to misexpress genes in a temporally and spatially controlled manner. However, this system suffers from two experimental limitations. First, the restriction enzyme-mediated integration procedure relies on chromosomal damage, resulting in a percentage of embryos failing to develop normally. Second, every transgenic embryo has unique sites of integration and unique transgene copy number, resulting in variable transgene expression levels and variable phenotypes. For these reasons, we have adapted the Gal4-UAS method for targeted gene expression to Xenopus. This technique relies on the generation of transgenic lines that carry "activator" or "effector" constructs. Activator lines express the yeast transcription factor, Gal4, under the control of a desired promoter, whereas effector lines contain DNA-binding motifs for Gal4-(UAS) linked to the gene of interest. We show that on intercrossing of these lines, the effector gene is transcribed in the temporal and spatial manner of the activator's promoter. Furthermore, we use the Gal4-UAS system to misexpress Xvent-2, a transcriptional target of bone morphogenetic protein 4 (BMP4) signaling during early embryogenesis. Embryos inheriting both the Gal4 activator and Xvent-2 effector transgenes display a consistent microcephalic phenotype. Finally, we exploit this system to characterize the neural and mesodermal defects obtained from early misexpression of Xvent-2. These results emphasize the potential of this system for the controlled analyses of gene function in Xenopus.

A Study of Mesoderm Patterning Through the Analysis of the Regulation of Xmyf-5 Expression

Development (Cambridge, England). Jun, 2002  |  Pubmed ID: 12050139

Xenopus laevis has been a particularly useful model organism for identifying factors involved in the induction and patterning of the mesoderm, however, much remains to be learned about how these factors interact. The myogenic transcription factor Xmyf-5 is the earliest known gene to be expressed specifically in the dorsolateral mesoderm of the gastrula, a domain that is established by the interaction of dorsal and ventral signals. For this reason, we have begun to investigate how the expression of Xmyf-5 is regulated. We have identified a 7.28 kb Xenopus tropicalis Xmyf-5 (Xtmyf-5) genomic DNA fragment that accurately recapitulates the expression of the endogenous gene. Deletion and mutational analysis has identified HBX2, an essential element, approximately 1.2 kb upstream from the start of transcription, which is necessary for both activation and repression of Xtmyf-5 expression, implying that positional information is integrated at this site. Electrophoretic mobility shift assays demonstrate that HBX2 specifically interacts with gastrula stage embryonic extracts and that in vitro translated Xvent-1 protein binds to one of its functional motifs. Combined with gain- and loss-of-function experiments, the promoter analysis described here suggests that Xvent-1 functions to repress Xmyf-5 expression in the ventral domain of the marginal zone. Furthermore, the identification of HBX2 provides a tool with which to identify other molecules involved in the regulation of Xmyf-5 expression during gastrulation.

Techniques and Probes for the Study of Xenopus Tropicalis Development

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Dec, 2002  |  Pubmed ID: 12454926

The frog Xenopus laevis has provided significant insights into developmental and cellular processes. However, X. laevis has an allotetraploid genome precluding its use in forward genetic analysis. Genetic analysis may be applicable to Xenopus (Silurana) tropicalis, which has a diploid genome and a shorter generation time. Here, we show that many tools for the study of X. laevis development can be applied to X. tropicalis. By using the developmental staging system of Nieuwkoop and Faber, we find that X. tropicalis embryos develop at similar rates to X. laevis, although they tolerate a narrower range of temperatures. We also show that many of the analytical reagents available for X. laevis can be effectively transferred to X. tropicalis. The X. laevis protocol for whole-mount in situ hybridization to mRNA transcripts can be successfully applied to X. tropicalis without alteration. Additionally, X. laevis probes often work in X. tropicalis--alleviating the immediate need to clone the X. tropicalis orthologs before initiating developmental studies. Antibodies that react against X. laevis proteins can effectively detect the X. tropicalis protein by using established immunohistochemistry procedures. Antisense morpholino oligonucleotides (MOs) offer a new alternative to study loss of gene activity during development. We show that MOs function in X. tropicalis. Finally, X. tropicalis offers the possibility for forward genetics and genomic analysis.

Molecular Components of the Endoderm Specification Pathway in Xenopus Tropicalis

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Jan, 2003  |  Pubmed ID: 12508233

Xenopus laevis has been instrumental in elucidating a conserved molecular pathway that regulates vertebrate endoderm specification. However, loss-of-function analysis is required to resolve the precise function of the genes involved. For such analysis, antisense oligos and possibly forward genetics are likely to be more effective in the diploid species Xenopus tropicalis than in the pseudotetraploid Xenopus laevis. Here we have isolated most of the tropicalis genes in the endoderm specification pathway, specifically, tVegT, tMixer, tMix, tBix, tGata6, tSox17alpha, tSox17beta, tFoxA1, tHex, and tCerberus, which lack the redundant copies that are found in laevis. In situ hybridization analysis has revealed identical expression patterns between the orthologous tropicalis and laevis endoderm genes, thus suggesting conserved genetic functions. Furthermore, we noted that the smaller tropicalis embryos gave better probe penetration than in laevis whole-mount in situ hybridizations-allowing us to visualize transcripts in the deep endoderm in tropicalis, which is difficult in laevis. This study illustrates how an entire genetic pathway can be quickly transferred from laevis to tropicalis due to high sequence conservation between the sister species and the large number of tropicalis-expressed sequence tags that are now available.

Local Tissue Interactions Across the Dorsal Midline of the Forebrain Establish CNS Laterality

Neuron. Jul, 2003  |  Pubmed ID: 12895418

The mechanisms that establish behavioral, cognitive, and neuroanatomical asymmetries are poorly understood. In this study, we analyze the events that regulate development of asymmetric nuclei in the dorsal forebrain. The unilateral parapineal organ has a bilateral origin, and some parapineal precursors migrate across the midline to form this left-sided nucleus. The parapineal subsequently innervates the left habenula, which derives from ventral epithalamic cells adjacent to the parapineal precursors. Ablation of cells in the left ventral epithalamus can reverse laterality in wild-type embryos and impose the direction of CNS asymmetry in embryos in which laterality is usually randomized. Unilateral modulation of Nodal activity by Lefty1 can also impose the direction of CNS laterality in embryos with bilateral expression of Nodal pathway genes. From these data, we propose that laterality is determined by a competitive interaction between the left and right epithalamus and that Nodal signaling biases the outcome of this competition.

Novel Gene Expression Domains Reveal Early Patterning of the Xenopus Endoderm

Gene Expression Patterns : GEP. Aug, 2003  |  Pubmed ID: 12915320

The endoderm gives rise the respiratory and digestive tract epithelia as well as associated organs such as the liver, lungs and pancreas. Investigations examining the molecular basis of embryonic endodermal patterning and organogenesis have been hampered by the lack of regionally expressed molecular markers in the early endoderm. By differentially screening an arrayed cDNA library, combined with an in situ hybridization screen we identified 13 new genes regionally expressed in the early tailbud endoderm of the Xenopus embryo. The putative proteins encoded by these cDNAs include a cell surface transporter, secreted proteins, a protease, a protease inhibitor, an RNA-binding protein, a phosphatase inhibitor and several enzymes. We find that the expression of these genes falls into one of three re-occurring domains in the tailbud embryo; (1). a ventral midgut, (2). posterior to the midgut and (3). in the dorsal endoderm beneath the notochord. Several of these genes are also regionally expressed at gastrula and neurula stages and appear to mark territories that were previously only predicted by the endoderm fate map. This indicates that there is significant positional identity in the early endoderm long before stages 28-32 when regional specification of the endoderm is thought to occur. These new genes provide valuable tools for studying endodermal patterning and organogenesis in Xenopus.

Pilot Morpholino Screen in Xenopus Tropicalis Identifies a Novel Gene Involved in Head Development

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Feb, 2004  |  Pubmed ID: 14745953

The diploid frog X. tropicalis has recently been adopted as a model genetic system, but loss-of-function screens in Xenopus have not yet been performed. We have undertaken a pilot functional knockdown screen in X. tropicalis for genes involved in nervous system development by injecting antisense morpholino (MO) oligos directed against X. tropicalis mRNAs. Twenty-six genes with primary expression in the nervous system were selected as targets based on an expression screen previously conducted in X. laevis. Reproducible phenotypes were observed for six and for four of these, a second MO gave a similar result. One of these genes encodes a novel protein with previously unknown function. Knocking down this gene, designated pinhead, results in severe microcephaly, whereas, overexpression results in macrocephaly. Together with the early embryonic expression in the anterior neural plate, these data indicate that pinhead is a novel gene involved in controlling head development.

Defining a Large Set of Full-length Clones from a Xenopus Tropicalis EST Project

Developmental Biology. Jul, 2004  |  Pubmed ID: 15223350

Amphibian embryos from the genus Xenopus are among the best species for understanding early vertebrate development and for studying basic cell biological processes. Xenopus, and in particular the diploid Xenopus tropicalis, is also ideal for functional genomics. Understanding the behavior of genes in this accessible model system will have a significant and beneficial impact on the understanding of similar genes in other vertebrate systems. Here we describe the analysis of 219,270 X. tropicalis expressed sequence tags (ESTs) from four early developmental stages. From these, we have deduced a set of unique expressed sequences comprising approximately 20,000 clusters and 16,000 singletons. Furthermore, we developed a computational method to identify clones that contain the complete coding sequence and describe the creation for the first time of a set of approximately 7000 such clones, the full-length (FL) clone set. The entire EST set is cloned in a eukaryotic expression vector and is flanked by bacteriophage promoters for in vitro transcription, allowing functional experiments to be carried out without further subcloning. We have created a publicly available database containing the FL clone set and related clustering data (http://www.gurdon.cam.ac.uk/informatics/Xenopus.html) and we make the FL clone set publicly available as a resource to accelerate the process of gene discovery and function in this model organism. The creation of the unique set of expressed sequences and the FL clone set pave the way toward a large-scale systematic analysis of gene sequence, gene expression, and gene function in this vertebrate species.

1.15 A Crystal Structure of the X. Tropicalis Spred1 EVH1 Domain Suggests a Fourth Distinct Peptide-binding Mechanism Within the EVH1 Family

FEBS Letters. Feb, 2005  |  Pubmed ID: 15710406

The recently described Spred protein family has been implicated in the modulation of receptor tyrosine kinase signalling. We report the crystal structure of the Enabled/vasodilator-stimulated phosphoprotein homology-1 (EVH1) domain from Xenopus tropicalis Spred1, solved to 1.15 A resolution. This structure confirms that the Spred EVH1 adopts the pleckstrin-homology fold, with a similar secondary structure to Enabled. A translation of one of the peptide-binding groove beta-strands narrows this groove, whilst one end of the groove shows structural flexibility. We propose that Spred1 will bind peptides that are less proline-rich than other EVH1 domains, with conformational changes indicating an induced fit.

Expression Cloning Screening of a Unique and Full-length Set of CDNA Clones is an Efficient Method for Identifying Genes Involved in Xenopus Neurogenesis

Mechanisms of Development. Mar, 2005  |  Pubmed ID: 15763209

Functional screens, where a large numbers of cDNA clones are assayed for certain biological activity, are a useful tool in elucidating gene function. In Xenopus, gain of function screens are performed by pool screening, whereby RNA transcribed in vitro from groups of cDNA clones, ranging from thousands to a hundred, are injected into early embryos. Once an activity is detected in a pool, the active clone is identified by sib-selection. Such screens are intrinsically biased towards potent genes, whose RNA is active at low quantities. To improve the sensitivity and efficiency of a gain of function screen we have bioinformatically processed an arrayed and EST sequenced set of 100,000 gastrula and neurula cDNA clones, to create a unique and full-length set of approximately 2500 clones. Reducing the redundancy and excluding truncated clones from the starting clone set reduced the total number of clones to be screened, in turn allowing us to reduce the pool size to just eight clones per pool. We report that the efficiency of screening this clone set is five-fold higher compared to a redundant set derived from the same libraries. We have screened 960 cDNA clones from this set, for genes that are involved in neurogenesis. We describe the overexpression phenotypes of 18 single clones, the majority of which show a previously uncharacterised phenotype and some of which are completely novel. In situ hybridisation analysis shows that a large number of these genes are specifically expressed in neural tissue. These results demonstrate the effectiveness of a unique full-length set of cDNA clones for uncovering players in a developmental pathway.

Identification of Novel Genes Affecting Mesoderm Formation and Morphogenesis Through an Enhanced Large Scale Functional Screen in Xenopus

Mechanisms of Development. Mar, 2005  |  Pubmed ID: 15763210

The formation of mesoderm is an important developmental process of vertebrate embryos, which can be broken down into several steps; mesoderm induction, patterning, morphogenesis and differentiation. Although mesoderm formation in Xenopus has been intensively studied, much remains to be learned about the molecular events responsible for each of these steps. Furthermore, the interplay between mesoderm induction, patterning and morphogenesis remains obscure. Here, we describe an enhanced functional screen in Xenopus designed for large-scale identification of genes controlling mesoderm formation. In order to improve the efficiency of the screen, we used a Xenopus tropicalis unique set of cDNAs, highly enriched in full-length clones. The screening strategy incorporates two mesodermal markers, Xbra and Xmyf-5, to assay for cell fate specification and patterning, respectively. In addition we looked for phenotypes that would suggest effects in morphogenesis, such as gastrulation defects and shortened anterior-posterior axis. Out of 1728 full-length clones we isolated 82 for their ability to alter the phenotype of tadpoles and/or the expression of Xbra and Xmyf-5. Many of the clones gave rise to similar misexpression phenotypes (synphenotypes) and many of the genes within each synphenotype group appeared to be involved in similar pathways. We determined the expression pattern of the 82 genes and found that most of the genes were regionalized and expressed in mesoderm. We expect that many of the genes identified in this screen will be important in mesoderm formation.

A Xenopus Tropicalis Oligonucleotide Microarray Works Across Species Using RNA from Xenopus Laevis

Mechanisms of Development. Mar, 2005  |  Pubmed ID: 15763212

Microarrays have great potential for the study of developmental biology. As a model system Xenopus is well suited for making the most of this potential. However, Xenopus laevis has undergone a genome wide duplication meaning that most genes are represented by two paralogues. This causes a number of problems. Most importantly the presence of duplicated genes mean that a X. laevis microarray will have less or even half the coverage of a similar sized microarray from the closely related but diploid frog Xenopus tropicalis. However, to date, X. laevis is the most commonly used amphibian system for experimental embryology. Therefore, we have tested if a microarray based on sequences from X. tropicalis will work across species using RNA from X. laevis. We produced a pilot oligonucleotide microarray based on sequences from X. tropicalis. The microarray was used to identify genes whose expression levels changed during early X. tropicalis development. The same assay was then carried out using RNA from X. laevis. The cross species experiments gave similar results to those using X. tropicalis RNA. This was true at the whole microarray level and for individual genes, with most genes giving similar results using RNA from X. laevis and X. tropicalis. Furthermore, the overlap in genes identified between a X. laevis and a X. tropicalis set of experiments was only 12% less than the overlap between two sets of X. tropicalis experiments. Therefore researchers can work with X. laevis and still make use of the advantages offered by X. tropicalis microarrays.

FGF Signal Interpretation is Directed by Sprouty and Spred Proteins During Mesoderm Formation

Developmental Cell. May, 2005  |  Pubmed ID: 15866160

Vertebrate gastrulation requires coordination of mesoderm specification with morphogenetic movements. While both of these processes require FGF signaling, it is not known how mesoderm specification and cell movements are coordinated during gastrulation. The related Sprouty and Spred protein families are recently discovered regulators of receptor tyrosine kinase signaling. We identified two genes for each family in Xenopus tropicalis: Xtsprouty1, Xtsprouty2, Xtspred1, and Xtspred2. In gain- and loss-of-function experiments we show that XtSprouty and XtSpred proteins modulate different signaling pathways downstream of the FGF receptor (FGFR), and consequently different developmental processes. Notably, XtSproutys inhibit morphogenesis and Ca(2+) and PKCdelta signaling, leaving MAPK activation and mesoderm specification intact. In contrast, XtSpreds inhibit MAPK activation and mesoderm specification, with little effect on Ca(2+) or PKCdelta signaling. These differences, combined with the timing of their developmental expression, suggest a mechanism to switch FGFR signal interpretation to coordinate mesoderm formation and cell movements during gastrulation.

Xenomics

Genome Research. Dec, 2005  |  Pubmed ID: 16339366

Xenopus genomics, or Xenomics for short, is coming of age. Indeed, biological insight into processes such as growth factor signaling and patterning of the early embryo is now being gained by combining the value of Xenopus as a model organism for cell and developmental biology with genomic approaches. In this review I address these recent advances and explore future possibilities gained from combining this powerful experimental system with genomic approaches, as well as how our quest to understand basic biological principles will be greatly facilitated though the marriage of Xenopus and genomics.

Maintenance of Motor Neuron Progenitors in Xenopus Requires a Novel Localized Cyclin

EMBO Reports. Mar, 2007  |  Pubmed ID: 17304238

The ventral spinal cord contains a pool of motor neuron progenitors (pMNs), which sequentially generate motor neurons and oligodendrocytes in the embryo. The mechanisms responsible for the maintenance of pMNs are not clearly understood. We have identified a novel cyclin, cyclin Dx (ccndx), which is specifically expressed in pMNs in Xenopus. Here, we show that inhibition of ccndx causes paralysis in embryos. Furthermore, we show that maintenance of pMNs requires ccndx function. In addition, inhibition of ccndx results in the specific loss of differentiated motor neurons. However, the expression of interneuron or sensory neuron markers is unaffected in these embryos, suggesting that the role of ccndx is specifically to maintain pMNs. Thus, we have identified, for the first time, a tissue-specific cell-cycle regulator that is essential for the maintenance of a pool of neural progenitors in the vertebrate spinal cord.

BDNF Promotes Target Innervation of Xenopus Mandibular Trigeminal Axons in Vivo

BMC Developmental Biology. 2007  |  Pubmed ID: 17540021

Trigeminal nerves consist of ophthalmic, maxillary, and mandibular branches that project to distinct regions of the facial epidermis. In Xenopus embryos, the mandibular branch of the trigeminal nerve extends toward and innervates the cement gland in the anterior facial epithelium. The cement gland has previously been proposed to provide a short-range chemoattractive signal to promote target innervation by mandibular trigeminal axons. Brain derived neurotrophic factor, BDNF is known to stimulate axon outgrowth and branching. The goal of this study is to determine whether BDNF functions as the proposed target recognition signal in the Xenopus cement gland.

Generation of Transgenic Xenopus Laevis: I. High-Speed Preparation of Egg Extracts

CSH Protocols. 2007  |  Pubmed ID: 21357171

INTRODUCTIONManipulating genes specifically during later stages of amphibian embryonic development requires fine control over the time and place of expression. These protocols describe an efficient nuclear-transplantation-based method of transgenesis developed for Xenopus laevis. The approach enables stable expression of cloned gene products in Xenopus embryos. Because the transgene integrates into the genome prior to fertilization, the resulting embryos are not chimeric, eliminating the need to breed to the next generation to obtain nonmosaic transgenic animals. The procedure is based on restriction-enzyme-mediated integration (REMI) and can be divided into three parts: (I) high-speed preparation of egg extracts, (II) sperm nuclei preparation, and (III) nuclear transplantation. This protocol describes the method for the high-speed preparation of egg extracts. Briefly, a crude, cytostatic factor (CSF)-arrested egg extract (i.e., cytoplasm arrested in meiotic metaphase) is prepared. These extracts are driven into the interphase stage of the cell cycle by addition of calcium, and high-speed centrifugation is performed to obtain a purer cytoplasmic fraction. This fraction promotes swelling of sperm nuclei, but does not promote DNA replication. By adding the egg extract to the reaction, the sperm chromatin partially decondenses, facilitating integration of plasmid DNA into the genome.

Generation of Transgenic Xenopus Laevis: II. Sperm Nuclei Preparation

CSH Protocols. 2007  |  Pubmed ID: 21357172

INTRODUCTIONManipulating genes specifically during later stages of amphibian embryonic development requires fine control over the time and place of expression. These protocols describe an efficient nuclear-transplantation-based method of transgenesis developed for Xenopus laevis. The approach enables stable expression of cloned gene products in Xenopus embryos. Because the transgene integrates into the genome prior to fertilization, the resulting embryos are not chimeric, eliminating the need to breed to the next generation to obtain nonmosaic transgenic animals. The procedure is based on restriction-enzyme-mediated integration (REMI) and can be divided into three parts: (I) high-speed preparation of egg extracts, (II) sperm nuclei preparation, and (III) nuclear transplantation. This protocol describes a method for the preparation of sperm nuclei from Xenopus laevis. Sperm suspensions are prepared by filtration and centrifugation, and then treated with lysolecithin to disrupt the plasma membrane of the cells. Sperm nuclei can be stored frozen in small aliquots at -80°C.

Generation of Transgenic Xenopus Laevis: III. Sperm Nuclear Transplantation

CSH Protocols. 2007  |  Pubmed ID: 21357173

INTRODUCTIONManipulating genes specifically during later stages of amphibian embryonic development requires fine control over the time and place of expression. These protocols describe an efficient nuclear-transplantation-based method of transgenesis developed for Xenopus laevis. The approach enables stable expression of cloned gene products in Xenopus embryos. The procedure is based on restriction-enzyme-mediated integration (REMI) and can be divided into three parts: (I) high-speed preparation of egg extracts, (II) sperm nuclei preparation, and (III) nuclear transplantation. This protocol describes a method for the nuclear transplantation in Xenopus laevis. Permeabilized sperm nuclei are incubated briefly with linearized plasmid DNA, after which egg extract and a small amount of restriction enzyme are added. The egg extract partially decondenses the chromosomes, and the restriction enzyme stimulates recombination by creating double-strand breaks, facilitating integration of DNA into the genome. Diluted nuclei are transplanted into unfertilized eggs. Because the transgene integrates into the genome prior to fertilization, the resulting transgenic embryos are not chimeric and there is no need to breed to the next generation in order to obtain nonmosaic transgenic animals.

Spib is Required for Primitive Myeloid Development in Xenopus

Blood. Sep, 2008  |  Pubmed ID: 18594023

Vertebrate blood formation occurs in 2 spatially and temporally distinct waves, so-called primitive and definitive hematopoiesis. Although definitive hematopoiesis has been extensively studied, the development of primitive myeloid blood has received far less attention. In Xenopus, primitive myeloid cells originate in the anterior ventral blood islands, the equivalent of the mammalian yolk sac, and migrate out to colonize the embryo. Using fluorescence time-lapse video microscopy, we recorded the migratory behavior of primitive myeloid cells from their birth. We show that these cells are the first blood cells to differentiate in the embryo and that they are efficiently recruited to embryonic wounds, well before the establishment of a functional vasculature. Furthermore, we isolated spib, an ETS transcription factor, specifically expressed in primitive myeloid precursors. Using spib antisense morpholino knockdown experiments, we show that spib is required for myeloid specification, and, in its absence, primitive myeloid cells retain hemangioblast-like characteristics and fail to migrate. Thus, we conclude that spib sits at the top of the known genetic hierarchy that leads to the specification of primitive myeloid cells in amphibians.

A Method for Generating Transgenic Frog Embryos

Methods in Molecular Biology (Clifton, N.J.). 2008  |  Pubmed ID: 19030816

Temporal and Spatial Expression of FGF Ligands and Receptors During Xenopus Development

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Jun, 2009  |  Pubmed ID: 19322767

Fibroblast growth factor (FGF) signalling plays a major role during early vertebrate development. It is involved in the specification of the mesoderm, control of morphogenetic movements, patterning of the anterior-posterior axis, and neural induction. In mammals, 22 FGF ligands have been identified, which can be grouped into seven subfamilies according to their sequence homology and function. We have cloned 17 fgf genes from Xenopus tropicalis and have analysed their temporal expression by RT-PCR and spatial expression by whole mount in situ hybridisation at key developmental stages. It reveals the diverse expression pattern of fgf genes during early embryonic development. Furthermore, our analysis shows the transient nature of expression of several fgfs in a number of embryonic tissues. This study constitutes the most comprehensive description of the temporal and spatial expression pattern of fgf ligands and receptors during vertebrate development to date. Developmental Dynamics 238:1467-1479, 2009. (c) 2009 Wiley-Liss, Inc.

C/EBPalpha Initiates Primitive Myelopoiesis in Pluripotent Embryonic Cells

Blood. Jul, 2009  |  Pubmed ID: 19420355

The molecular mechanisms that underlie the development of primitive myeloid cells in vertebrate embryos are not well understood. Here we characterize the role of cebpa during primitive myeloid cell development in Xenopus. We show that cebpa is one of the first known hematopoietic genes expressed in the embryo. Loss- and gain-of-function studies show that it is both necessary and sufficient for the development of functional myeloid cells. In addition, we show that cebpa misexpression leads to the precocious induction of myeloid cell markers in pluripotent prospective ectodermal cells, without the cells transitioning through a general mesodermal state. Finally, we use live imaging to show that cebpa-expressing cells exhibit many attributes of terminally differentiated myeloid cells, such as highly active migratory behavior, the ability to quickly and efficiently migrate toward wounds and phagocytose bacteria, and the ability to enter the circulation. Thus, C/EPBalpha is the first known single factor capable of initiating an entire myelopoiesis pathway in pluripotent cells in the embryo.

Germ Layer Specification and Axial Patterning in the Embryonic Development of the Freshwater Planarian Schmidtea Polychroa

Developmental Biology. Apr, 2010  |  Pubmed ID: 20100474

Although patterning during regeneration in adult planarians has been studied extensively, very little is known about how the initial planarian body plan arises during embryogenesis. Herein, we analyze the process of embryo patterning in the species Schmidtea polychroa by comparing the expression of genes involved in the establishment of the metazoan body plan. Planarians present a derived ectolecithic spiralian development characterized by dispersed cleavage within a yolk syncytium and an early transient embryo capable of feeding on the maternally supplied yolk cells. During this stage of development, we only found evidence of canonical Wnt pathway, mostly associated with the development of its transient pharynx. At these stages, genes involved in gastrulation (snail) and germ layer determination (foxA and twist) are specifically expressed in migrating blastomeres and those giving rise to the temporary gut and pharyngeal muscle. After yolk ingestion, the embryo expresses core components of the canonical Wnt pathway and the BMP pathway, suggesting that the definitive axial identities are established late. These data support the division of planarian development into two separate morphogenetic stages: a highly divergent gastrulation stage, which segregates the three germ layers and establishes the primary organization of the feeding embryo; and subsequent metamorphosis, based on totipotent blastomeres, which establishes the definitive adult body plan using mechanisms that are similar to those used during regeneration and homeostasis in the adult.

The Genome of the Western Clawed Frog Xenopus Tropicalis

Science (New York, N.Y.). Apr, 2010  |  Pubmed ID: 20431018

The western clawed frog Xenopus tropicalis is an important model for vertebrate development that combines experimental advantages of the African clawed frog Xenopus laevis with more tractable genetics. Here we present a draft genome sequence assembly of X. tropicalis. This genome encodes more than 20,000 protein-coding genes, including orthologs of at least 1700 human disease genes. Over 1 million expressed sequence tags validated the annotation. More than one-third of the genome consists of transposable elements, with unusually prevalent DNA transposons. Like that of other tetrapods, the genome of X. tropicalis contains gene deserts enriched for conserved noncoding elements. The genome exhibits substantial shared synteny with human and chicken over major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage.

Cerebellar Involvement in Hodgkin's Lymphoma: an Atypical Site of Relapse

Clinical & Translational Oncology : Official Publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico. Jun, 2010  |  Pubmed ID: 20534402

The presentation of intracranial metastases from Hodgkin's lymphoma is an infrequent event that worsens clinical outcome. A case of Hodgkin's lymphoma relapse in the cerebellum is described in a 70-year-old woman with a previously treated stage IVA Hodgkin's lymphoma. Diagnostic workup and treatment strategies for central nervous system relapses are reviewed and discussed. A combination of surgery, radiotherapy and occasionally chemotherapy remains the most appropriate approach to intracranial Hodgkin's lymphoma.

Cis-Regulatory Remodeling of the SCL Locus During Vertebrate Evolution

Molecular and Cellular Biology. Dec, 2010  |  Pubmed ID: 20956563

Development progresses through a sequence of cellular identities which are determined by the activities of networks of transcription factor genes. Alterations in cis-regulatory elements of these genes play a major role in evolutionary change, but little is known about the mechanisms responsible for maintaining conserved patterns of gene expression. We have studied the evolution of cis-regulatory mechanisms controlling the SCL gene, which encodes a key transcriptional regulator of blood, vasculature, and brain development and exhibits conserved function and pattern of expression throughout vertebrate evolution. SCL cis-regulatory elements are conserved between frog and chicken but accrued alterations at an accelerated rate between 310 and 200 million years ago, with subsequent fixation of a new cis-regulatory pattern at the beginning of the mammalian radiation. As a consequence, orthologous elements shared by mammals and lower vertebrates exhibit functional differences and binding site turnover between widely separated cis-regulatory modules. However, the net effect of these alterations is constancy of overall regulatory inputs and of expression pattern. Our data demonstrate remarkable cis-regulatory remodelling across the SCL locus and indicate that stable patterns of expression can mask extensive regulatory change. These insights illuminate our understanding of vertebrate evolution.

FGF Signalling: Diverse Roles During Early Vertebrate Embryogenesis

Development (Cambridge, England). Nov, 2010  |  Pubmed ID: 20978071

Fibroblast growth factor (FGF) signalling has been implicated during several phases of early embryogenesis, including the patterning of the embryonic axes, the induction and/or maintenance of several cell lineages and the coordination of morphogenetic movements. Here, we summarise our current understanding of the regulation and roles of FGF signalling during early vertebrate development.

Characterisation of a New Regulator of BDNF Signalling, Sprouty3, Involved in Axonal Morphogenesis in Vivo

Development (Cambridge, England). Dec, 2010  |  Pubmed ID: 21062861

During development, many organs, including the kidney, lung and mammary gland, need to branch in a regulated manner to be functional. Multicellular branching involves changes in cell shape, proliferation and migration. Axonal branching, however, is a unicellular process that is mediated by changes in cell shape alone and as such appears very different to multicellular branching. Sprouty (Spry) family members are well-characterised negative regulators of Receptor tyrosine kinase (RTK) signalling. Knockout of Spry1, 2 and 4 in mouse result in branching defects in different organs, indicating an important role of RTK signalling in controlling branching pattern. We report here that Spry3, a previously uncharacterised member of the Spry family plays a role in axonal branching. We found that spry3 is expressed specifically in the trigeminal nerve and in spinal motor and sensory neurons in a Brain-derived neurotrophin factor (BDNF)-dependent manner. Knockdown of Spry3 expression causes an excess of axonal branching in spinal cord motoneurons in vivo. Furthermore, Spry3 inhibits the ability of BDNF to induce filopodia in Xenopus spinal cord neurons. Biochemically, we show that Spry3 represses calcium release downstream of BDNF signalling. Altogether, we have found that Spry3 plays an important role in the regulation of axonal branching of motoneurons in vivo, raising the possibility of unexpected conservation in the involvement of intracellular regulators of RTK signalling in multicellular and unicellular branching.

Genome-wide Analysis of Gene Expression During Xenopus Tropicalis Tadpole Tail Regeneration

BMC Developmental Biology. 2011  |  Pubmed ID: 22085734

The molecular mechanisms governing vertebrate appendage regeneration remain poorly understood. Uncovering these mechanisms may lead to novel therapies aimed at alleviating human disfigurement and visible loss of function following injury. Here, we explore tadpole tail regeneration in Xenopus tropicalis, a diploid frog with a sequenced genome.

PTransgenesis: a Cross-species, Modular Transgenesis Resource

Development (Cambridge, England). Dec, 2011  |  Pubmed ID: 22110059

As studies aim increasingly to understand key, evolutionarily conserved properties of biological systems, the ability to move transgenesis experiments efficiently between organisms becomes essential. DNA constructions used in transgenesis usually contain four elements, including sequences that facilitate transgene genome integration, a selectable marker and promoter elements driving a coding gene. Linking these four elements in a DNA construction, however, can be a rate-limiting step in the design and creation of transgenic organisms. In order to expedite the construction process and to facilitate cross-species collaborations, we have incorporated the four common elements of transgenesis into a modular, recombination-based cloning system called pTransgenesis. Within this framework, we created a library of useful coding sequences, such as various fluorescent protein, Gal4, Cre-recombinase and dominant-negative receptor constructs, which are designed to be coupled to modular, species-compatible selectable markers, promoters and transgenesis facilitation sequences. Using pTransgenesis in Xenopus, we demonstrate Gal4-UAS binary expression, Cre-loxP-mediated fate-mapping and the establishment of novel, tissue-specific transgenic lines. Importantly, we show that the pTransgenesis resource is also compatible with transgenesis in Drosophila, zebrafish and mammalian cell models. Thus, the pTransgenesis resource fosters a cross-model standardization of commonly used transgenesis elements, streamlines DNA construct creation and facilitates collaboration between researchers working on different model organisms.