Biomedical subjects

Aaron M Zorn

Publications and source records attributed to Aaron M Zorn.

17 recordsLinked to original sources

PubMed2007

FoxI1e activates ectoderm formation and controls cell position in the Xenopus blastula.

The segregation of the vertebrate embryo into three primary germ layers is one of the earliest developmental decisions. In Xenopus, where the process is best understood, the endoderm is specified by a vegetally localized transcription factor, VegT, which releases nodal signals that specify the adjacent marginal zone of the blastula to become mesoderm. However, little is known about how the ectoderm becomes specified. In this paper, we show that the forkhead protein FoxI1e (also known as Xema) is required at the blastula stage for normal formation of both the central nervous system and epidermis, the two early derivatives of the ectoderm. In addition, FoxI1e is required to maintain the regional identity of the animal cells of the blastula, the cells that are precursors of ectodermal structures. In its absence, they lose contact with the animal cap, mix with cells of other germ layers and differentiate according to their new positions. Because FoxI1e is initially expressed in the animal region of the embryo and is rapidly downregulated in the neural plate, its role in neural and epidermal gene expression must precede the division of the ectoderm into neural and epidermal. The work also shows that FoxI1e plays a role in the embryo in the poorly understood process of differential adhesion, which limits cell mixing as primary germ layers become specified.

Animals↗

PubMed2006

Sox17 influences the differentiation of respiratory epithelial cells.

The Sry-related HMG box transcription factor, Sox17, is required for formation of definitive endoderm that gives rise to various organs, including thyroid, lung, liver, pancreas, and intestine. While expressed at high levels in the embryonic endoderm, Sox17 is also expressed in mature tissues, including the lung. Sox17 expression in respiratory epithelial cells was first detected in the fetal lung at embryonic day 18. Thereafter, Sox17 expression was restricted primarily to ciliated cells, suggesting its potential role in airway cell differentiation. When expressed in epithelial cells of the embryonic lung, Sox17 inhibited peripheral epithelial cell differentiation and disrupted branching morphogenesis. In vitro, Sox17 inhibited Sftpc and enhanced Foxj1 promoter activity, consistent with its expression in proximal airway cells. Conditional expression of Sox17 in peripheral respiratory epithelial cells of adult lung induced hyperplastic clusters of cells expressing increased levels of beta-catenin and differentiation markers representing multiple proximal respiratory epithelial cell types. Sox17 prolonged survival and enhanced growth and differentiation of respiratory epithelial cells in vitro. Sox17 induced plasticity of respiratory epithelial cells, reprogramming alveolar cells into epithelial cells with characteristics more typical of the proximal airway. Sites of expression and the effects of Sox17 in vivo and in vitro are consistent with a role for Sox17 or other members of the Sox family of transcription factors in differentiation of the conducting airway epithelium.

Animals↗

PubMed2006

A new method to remove hybridization bias for interspecies comparison of global gene expression profiles uncovers an association between mRNA sequence divergence and differential gene expression in Xenopus.

The recent sequencing of a large number of Xenopus tropicalis expressed sequences has allowed development of a high-throughput approach to study Xenopus global RNA gene expression. We examined the global gene expression similarities and differences between the historically significant Xenopus laevis model system and the increasingly used X.tropicalis model system and assessed whether an X.tropicalis microarray platform can be used for X.laevis. These closely related species were also used to investigate a more general question: is there an association between mRNA sequence divergence and differences in gene expression levels? We carried out a comprehensive comparison of global gene expression profiles using microarrays of different tissues and developmental stages of X.laevis and X.tropicalis. We (i) show that the X.tropicalis probes provide an efficacious microarray platform for X.laevis, (ii) describe methods to compare interspecies mRNA profiles that correct differences in hybridization efficiency and (iii) show independently of hybridization bias that as mRNA sequence divergence increases between X.laevis and X.tropicalis differences in mRNA expression levels also increase.

Animals↗

PubMed2006

Molecular control of liver development.

Recent studies using animal models have elucidated a growing number of evolutionarily conserved genes and pathways that control liver development from the embryonic endoderm. It is increasingly clear that the genetic programs active in embryogenesis are often deregulated or reactivated in disease, cancer, and tissue repair. Understanding the molecular control of liver development should impact diagnosis and treatment of pediatric and adult liver diseases and aid in efforts to differentiate liver tissue in vitro for stem cell-based therapies.

Animals↗

PubMed2006

Global analysis of the transcriptional network controlling Xenopus endoderm formation.

A conserved molecular pathway has emerged controlling endoderm formation in Xenopus zebrafish and mice. Key genes in this pathway include Nodal ligands and transcription factors of the Mix-like paired homeodomain class, Gata4-6 zinc-finger factors and Sox17 HMG domain proteins. Although a linear epistatic pathway has been proposed, the precise hierarchical relationships between these factors and their downstream targets are largely unresolved. Here, we have used a combination of microarray analysis and loss-of-function experiments to examine the global regulatory network controlling Xenopus endoderm formation. We identified over 300 transcripts enriched in the gastrula endoderm, including most of the known endoderm regulators and over a hundred uncharacterized genes. Surprisingly only 10% of the endoderm transcriptome is regulated as predicted by the current linear model. We find that Nodal genes, Mixer and Sox17 have both shared and distinct sets of downstream targets, and that a number of unexpected autoregulatory loops exist between Sox17 and Gata4-6, between Sox17 and Bix1/Bix2/Bix4, and between Sox17 and Xnr4. Furthermore, we find that Mixer does not function primarily via Sox17 as previously proposed. These data provides new insight into the complexity of endoderm formation and will serve as valuable resource for establishing a complete endoderm gene regulatory network.

Animals↗

PubMed2005

Transdifferentiation of ciliated cells during repair of the respiratory epithelium.

Since the lung is repeatedly subjected to injury by pathogens and toxicants, maintenance of pulmonary homeostasis requires rapid repair of its epithelial surfaces. Ciliated bronchiolar epithelial cells, previously considered as terminally differentiated, underwent squamous cell metaplasia within hours after bronchiolar injury with naphthalene. Expression of transcription factors active in morphogenesis and differentiation of the embryonic lung, including beta-catenin, Foxa2, Foxj1, and Sox family members (Sox17 and Sox2), was dynamically regulated during repair and redifferentiation of the bronchiolar epithelium after naphthalene injury. Squamous cells derived from ciliated cells spread beneath injured Clara cells within 6-12 h after injury, maintaining the integrity of the epithelium. Dynamic changes in cell shape and gene expression, indicating cell plasticity, accompanied the transition from squamous to cuboidal to columnar cell types as differentiation-specific cell markers typical of the mature airway were restored. Similar dynamic changes in the expression of these transcription factors occurred in ciliated and Clara cells during regeneration of the lung after unilateral pneumonectomy. Taken together, these findings demonstrate that ciliated epithelial cells spread and transdifferentiate into distinct epithelial cell types to repair the airway epithelium.

Animals↗

PubMed2005

Solution structure and backbone dynamics of the KH-QUA2 region of the Xenopus STAR/GSG quaking protein.

The Quaking protein belongs to the family of STAR/GSG domain RNA-binding proteins and is involved in multiple cell signalling and developmental processes in vertebrates, including the formation of myelin. Heteronuclear NMR methods were used to determine the solution structure of a 134 residue fragment spanning the KH and QUA2 homology regions of the Quaking protein from Xenopus laevis (pXqua) in the absence of RNA. The protein is shown to adopt an extended type I KH domain fold that is connected to a structured alpha-helix in the C-terminal QUA2 region by means of a highly flexible linker. A comparison with the solution structure of the related protein splicing factor 1 (SF1) indicates that most aspects of the RNA-binding interface are conserved in pXqua, although the "variable loop" region that follows the second beta-strand possesses two additional alpha-helices. The structure of pXqua provides an appropriate template for building models of important homologues, such as GLD-1 and Sam68. Measurements of the (15)N relaxation parameters of pXqua confirm that the polypeptide backbone of the QUA2 region is more dynamic than that of the KH portion, and that the C-terminal helix is partially structured in the absence of RNA. By comparison with a random coil reference state, the nascent structure in the QUA2 region is estimated to contribute 15.5kJmol(-1) to the change in conformational free energy that occurs on forming a complex with RNA. Since STAR/GSG proteins may regulate alternative splicing by competing with SF1 in the nucleus for specific branch-point sequences that signal intronic RNA, the formation of secondary structure in the QUA2 region in the unbound state of pXqua has important functional consequences.

Amino Acid Sequence↗

PubMed2004

Negative regulation of Smad2 by PIASy is required for proper Xenopus mesoderm formation.

Mesoderm induction and patterning are primarily regulated by the concentration of locally expressed morphogens such as members of the TGFbetasuperfamily. Smad2 functions as a transcription factor to regulate expression of mesodermal genes downstream of such morphogens. We have identified Xenopus PIASy (XPIASy), a member of the PIAS family, by yeast two-hybrid screening using Xenopus Smad2 (XSmad2) as a bait. During mesoderm induction, XPIASy is expressed in the animal half of embryos with a ventral high-dorsal low gradient at the marginal zone. XPIASy expression is positively and negatively regulated by activities of the XSmad2 and Wnt pathways, respectively. Interestingly, inhibition of XPIASy by morpholinos induces elongation of animal caps with induction of mesoderm genes even in the absence of their morphogen-mediated activation. In addition, their introduction into the ventral marginal zone results in a secondary axis formation. Gain-of-function analysis revealed that XPIASy inhibits mesoderm induction by specific and direct downregulation of XSmad2 transcriptional activity. These observations indicate that XPIASy functions as an essential negative regulator of the XSmad2 pathway to ensure proper mesoderm induction at the appropriate time and in the appropriate region, and suggest that both the initial step of morphogen-mediated activation of the XSmad2 pathway and regulation of the final downstream transcription step have crucial roles in mesoderm induction and patterning.

Amino Acid Sequence↗

PubMed2004

Defining a large set of full-length clones from a Xenopus tropicalis EST project.

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.

Animals↗

PubMed2004

Smad2 and Smad3 coordinately regulate craniofacial and endodermal development.

Ligands of the transforming growth factor-beta (TGF-beta) superfamily are involved in numerous developmental and disease processes. TGF-beta, activins, and nodal ligands operate through the highly homologous Smad2 and Smad3 intracellular mediators. Smad2 mutants exhibit early embryonic lethality, while Smad3 mutants are viable, but show a plethora of postnatal phenotypes, including immune dysfunction and skeletal abnormalities. Previously, we have shown that the Smad2 and Smad3 genes function cooperatively during liver morphogenesis. Here we show that Smad2 and Smad3 are required at a full dosage for normal embryonic development. Animals lacking one allele of each gene exhibit a variably penetrant phenotype in which structures in the anterior and ventral midline are reduced or lost; additionally, we demonstrate that this craniofacial defect and the previously reported hepatic phenotypes are both due to defects in the definitive endoderm. A reduction of endodermal gene expression as well as a failure to displace the visceral endoderm occurs despite the formation of a normal foregut pocket. This precedes any defects in anterior patterning and likely causes the abnormalities observed in craniofacial and midline development, as well as hepatogenesis.

Animals↗

PubMed2004

Sox17 and beta-catenin cooperate to regulate the transcription of endodermal genes.

Recent studies have led to a model of the molecular pathway that specifies the endoderm during vertebrate gastrulation. The HMG box transcription factor Sox17 is a key component of this pathway and is essential for endoderm formation; however, the molecular events controlled by Sox17 are largely unknown. We have identified several direct transcriptional targets of Sox17, including Foxa1 and Foxa2. We show that beta-catenin, a component of Wnt signaling pathway, physically interacts with Sox17 and potentiates its transcriptional activation of target genes. We identify a motif in the C terminus of Sox17, which is conserved in all the SoxF subfamily of Sox proteins, and this motif is required for the ability of Sox17 to both transactivate target genes and bind beta-catenin. Nuclear beta-catenin is present in endoderm cells of the gastrula, and depletion of beta-catenin from embryos results in a repression of Sox17 target genes. These data suggest that in a mechanism analogous to Tcf/Lef interacting with beta-catenin, Sox17 and beta-catenin interact to transcribe endodermal target genes.

Amino Acid Motifs↗

PubMed2003

Cardiac T-box factor Tbx20 directly interacts with Nkx2-5, GATA4, and GATA5 in regulation of gene expression in the developing heart.

Tbx20 is a member of the T-box transcription factor family expressed in the forming hearts of vertebrate and invertebrate embryos. We report here analysis of Tbx20 expression during murine cardiac development and assessment of DNA-binding and transcriptional properties of Tbx20 isoforms. Tbx20 was expressed in myocardium and endocardium, including high levels in endocardial cushions. cDNAs generated by alternative splicing encode at least four Tbx20 isoforms, and Tbx20a uniquely carried strong transactivation and transrepression domains in its C terminus. Isoforms with an intact T-box bound specifically to DNA sites resembling the consensus brachyury half site, although with less avidity compared with the related factor, Tbx5. Tbx20 physically interacted with cardiac transcription factors Nkx2-5, GATA4, and GATA5, collaborating to synergistically activate cardiac gene expression. Among cardiac GATA factors, there was preferential synergy with GATA5, implicated in endocardial differentiation. In Xenopus embryos, enforced expression of Tbx20a, but not Tbx20b, led to induction of mesodermal and endodermal lineage markers as well as cell migration, indicating that the long Tbx20a isoform uniquely bears functional domains that can alter gene expression and developmental behaviour in an in vivo context. We propose that Tbx20 plays an integrated role in the ancient myogenic program of the heart, and has been additionally coopted during evolution of vertebrates for endocardial cushion development.

Alternative Splicing↗

PubMed2003

Organogenesis: making pancreas from liver.

Making endocrine pancreas cells at will is one of the major goals of cellular based therapies for diabetes. The experimentally induced conversion of hepatocytes into pancreatic cells, using a modified version of the transcription factor Pdx-1, may provide an alternative to stem cell approaches.

Animals↗

PubMed2003

Molecular components of the endoderm specification pathway in Xenopus tropicalis.

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.

Animals↗

PubMed2003

Novel gene expression domains reveal early patterning of the Xenopus endoderm.

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.

Animals↗

PubMed2002

Techniques and probes for the study of Xenopus tropicalis development.

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.

Animals↗

PubMed2002

A dynamic requirement for community interactions during Xenopus myogenesis.

The community effect is an interaction among a group of many nearby cells that is necessary for them to maintain tissue-specific gene expression and differentiate co-ordinately. A community interaction is required for the muscle precursor cells of the Xenopus embryo to develop into terminally differentiated muscle, but exactly when and where the community effect acts during myogenesis has not been determined. Here, we ask whether dependence on the community effect varies with the developmental age of the muscle precursor cells. We find that dependence on the community signal changes with time through the muscle precursor cell population. During neurulation muscle precursor cells that are still in the vicinity of the blastopore and that are fated to form posterior muscle continue to require interactions with their neighbours, while differentiation of the anterior paraxial mesoderm,which gastrulated earlier, is independent of cell contact at this time. Thus the time during which a particular sub-population of muscle precursor cells requires a community interaction is related to their final destination along the anterior-posterior axis. In addition we show that this later acting community interaction around the blastopore involves FGF signalling.

Animals↗